The Complement System: Innovative Diagnostic and Research Protocols (Methods in Molecular Biology, 2227) 1071610155, 9781071610152

Table of contents :
Preface
Contents
Contributors
Chapter 1: The Benefits of Complement Measurements for the Clinical Practice
1 Complement system in Physiology
2 Role of Complement in Pathology
3 Complement as a Drug Target
4 Challenges for Complement Monitoring
References
Chapter 2: Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source
1 Introduction
2 Materials
2.1 IgG and IgM Depletion by FLPC
2.2 ELISA
2.3 Hemolytic Assays
3 Methods
3.1 IgG and IgM Depletion by Affinity Chromatography
3.2 Recovery of Bound IgG and IgM from Column
3.3 IgG ELISA
3.4 IgM ELISA
3.5 C1q ELISA
3.6 Classical Pathway Hemolytic Assay (CH50)
3.7 Alternative Pathway Hemolytic Assay (AH50)
4 Notes
References
Chapter 3: Quantification of Complement Proteins with Special Reference to C1q: Multiplex Versus ELISA Versus Rocket Immunoele...
1 Introduction
2 Material
2.1 Coupled Beads
2.2 Buffers and Reagents
2.3 Antibodies
2.4 Calibration, Controls, and Samples
3 Method
4 Notes
References
Chapter 4: C3dg Quantification by PEG Precipitation and or TRIFMA
1 Introduction
2 Materials
3 Methods
3.1 C3dg Quantification by Precipitation and ELISA
3.2 C3dg Quantification by PEG Precipitation and Time-Resolved Immunofluorometric Assay (TRIFMA) (See Note 3)
4 Notes
References
Chapter 5: Quantification of Porcine Complement Activation Fragment C3a by a Neoepitope-Based Enzyme-Linked Immunosorbent Assay
1 Introduction
2 Materials
2.1 Buffer Preparation
2.2 Working Solutions
2.3 Antibodies
2.4 Samples, Standards, and Controls
2.5 Cross-Reactivity, Spiking, and Coefficients of Variation
2.6 Example of Usage-Detection of C3a in In Vivo Samples from Porcine Sepsis
3 Methods
4 Notes
References
Chapter 6: Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities
1 Introduction
2 Materials
2.1 Preparation of Sensitized Sheep Erythrocytes
2.1.1 Common Supplies
2.1.2 Buffers
2.1.3 Biological Components
2.2 Preparation of C3b-Sensitized Sheep Erythrocytes
2.2.1 Common Supplies
2.2.2 Buffers
2.2.3 Biological Compounds
3 Methods
3.1 Preparation of Sheep Erythrocytes (See Note 1)
3.2 Preparation of Sensitized SE (See Note 3)
3.3 Preparation of C3b-Coated SE Suspension (See Note 5)
3.3.1 Preparation of Zymosan-Activated Human Serum (ZASH)
3.3.2 Preparation of C3b-Coated Cell Suspension
4 Notes
References
Chapter 7: Hemolytic Tests Exploring Factor H Functional Activities
1 Introduction
2 Materials
2.1 Materials for the Hemolytic Assay Studying the AP Decay Activity of FH
2.1.1 Common Supplies
2.1.2 Buffers
2.1.3 Biological Components
2.2 Materials for the Hemolytic Assay Studying the FH Activity on Cell Surface Protection
2.2.1 Common Supplies
2.2.2 Buffers
2.2.3 Biological Components
3 Methods
3.1 Preparation of C3bBb-Coated Sheep Erythrocytes (see Note 1)
3.1.1 Preparation of the C3bBb-Coated Cell Suspension
3.1.2 Determination of the Optimal Conditions
3.1.3 Calculations
3.2 Testing FH Decay Activity
3.2.1 Experimental Procedure per Sample to Be Analyzed (11 Tubes Are Needed per Sample) (Table 2)
3.2.2 Calculation (See Note 3, Table 3))
3.3 Hemolytic Assay Studying the FH Activity of Cell Surface Protection (See Note 5)
3.3.1 Preparation of Sheep Erythrocytes
3.3.2 Testing FH Activity on Cell Surface Protection
3.3.3 Calculation (See Note 6)
4 Notes
References
Chapter 8: Functional Hemolytic Test for Complement Alternative Pathway Convertase Activity
1 Introduction
2 Materials
2.1 Assay Buffers
2.2 Human Sera (See Note 5)
2.3 Other Reagents
2.4 Specific Laboratory Equipment
3 Methods
3.1 Washing of Rabbit Erythrocytes
3.2 Calibration of Erythrocyte Working Suspension
3.3 Preparation of serum Samples
3.4 Convertase Activity Assay-Step 1; Convertase Assembly
3.5 Convertase Activity Assay-Washing Step
3.6 Convertase Activity Assay-Step 2; Convertase Readout with Hemolysis
3.7 Data Analysis
3.8 Additional Research for Characterization
4 Notes
References
Chapter 9: Complement C3 Deposition on Endothelial Cells Revealed by Flow Cytometry
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 10: Anti-C1q Autoantibodies: Standard Quantification and Innovative ELISA
1 Introduction
2 Materials
2.1 Buffers
2.2 Antigens and Conjugates for C1q ELISA
2.3 Antigens and Conjugates for Anti-A08 of C1q
3 Methods
3.1 Method for Anti-C1q
3.2 Method for Anti-A08 of C1q
4 Notes
References
Chapter 11: Anti-C1-Inhibitor Autoantibody Detection by ELISA
1 Introduction
2 Materials
2.1 Blood Collection
2.2 ELISA
3 Methods
3.1 Blood Collection
3.2 Coating of the Plate
3.3 Samples Dilution
3.4 Incubation
3.5 Primary Antibody
3.6 Secondary Antibody
3.7 Analysis Step
4 Notes
References
Chapter 12: Anti-Ficolin-2 and Anti-Ficolin-3 Autoantibody Detection by ELISA
1 Introduction
2 Materials
2.1 Ficolin Production
2.2 Anti-ficolin Autoantibody ELISA
3 Methods
3.1 Ficolins Production
3.1.1 Production of Recombinant Ficolin-2
3.1.2 Production of Recombinant Ficolin-3
3.2 Anti-ficolin Autoantibody ELISA
3.2.1 Anti-Ficolin-2 Autoantibody ELISA
3.2.2 Anti-Ficolin-3 Autoantibody ELISA
3.3 Data Analysis
3.3.1 Anti-Ficolin-2 Autoantibody Analysis
3.3.2 Anti-Ficolin-3 Autoantibody Analysis
4 Notes
References
Chapter 13: Detection of Anti-C3b Autoantibodies by ELISA
1 Introduction
2 Materials
2.1 Collection of the Blood Samples
2.2 ELISA
3 Methods
4 Notes
References
Chapter 14: Detection of Complement Factor B Autoantibodies by ELISA
1 Introduction
2 Materials
3 Method
4 Notes
References
Chapter 15: Detection of C3 Nephritic Factor by Hemolytic Assay
1 Introduction
2 Materials
2.1 A-Step A: Preparation of Sheep Erythrocytes
2.1.1 Common Supplies
2.1.2 Buffers
2.1.3 Biological Components
2.2 B-Step B: Preparation of Sensitized Sheep Erythrocytes
2.2.1 Common Supplies
2.2.2 Buffers
2.2.3 Biological Components
2.3 Step C-Preparation of Sheep Erythrocytes Bearing C3b
2.3.1 Common Supplies
2.3.2 Buffers
2.3.3 Biological Components
2.4 Step D: Purification of Total IgG from Patient Plasma (See Note 1)
2.5 Step E-C3Nef Assays with Sheep Erythrocytes Bearing C3b
2.5.1 Common Supplies
2.5.2 Buffers
2.5.3 Biological Components
3 Methods
3.1 A-Step A: Preparation of Sheep Erythrocytes
3.2 B-Step B: Preparation of Sensitized Sheep Erythrocytes
3.3 C-Step C: Preparation of Sheep Erythrocytes Bearing C3b
3.3.1 Preparation of Zymozan-Activated Human Serum (ZAHS) Solution
3.4 Preparation of Sheep Erythrocytes Bearing C3b
3.5 Step D: Purification of Total IgG from Patient Plasma
3.6 Step E: C3Nef Assays with Sheep Erythrocytes Bearing C3b
3.6.1 Determine Concentration of Factor B (FB) to Obtain 1.5 Lytic Sites per Cell (Z = 1.5) (Fig. 1)
3.6.2 C3NeF Assay (Fig. 2)
3.7 Convertase Formation on Sheep Erythrocytes
3.7.1 Convertase Dissociation
3.7.2 Lysis
3.7.3 Calculation of the Percentage of Stabilization
4 Notes
References
Chapter 16: Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequ...
1 Introduction
1.1 CNVs in the CR1 Gene
1.2 CNVs in the CFH and CFHR1-5 Genes
1.3 MLPA and CNV Analysis from NGS Data
2 Materials
2.1 MLPA
2.2 CNV Analysis from NGS Data
2.2.1 NextGENe Software
2.2.2 ONCOCNV Software
3 Methods
3.1 MLPA Analysis
3.1.1 Preparation of DNA
3.1.2 MLPA Assay
DNA Denaturation and Hybridization of the Probes
Ligation
PCR Reaction
3.1.3 Analysis by Capillary Electrophoresis
3.1.4 Analysis by Coffalyser Software
Setting the Parameters for the Analysis
Analysis of .fsa Files Obtained After Capillary Electrophoresis
Interpretation of the Results (Ratio Chart)
3.2 Identification of CNVs in the Data Generated from NGS Gene Panels
3.2.1 NextGENe
Running the NextGENe CNV Tool
3.2.2 ONCOCNV
Running the ONCOCNV Tool
3.3 MLPA Versus Identification of CNVs in the Data Generated from NGS Gene Panels
3.3.1 Pros and Cons
4 Notes
References
Chapter 17: Complement Detection in Mouse Kidneys by Immunofluorescence
1 Introduction
2 Materials
2.1 Frozen and FFPE Tissues
2.2 Frozen Tissues Only
2.3 FFPE Tissues Only
3 Methods
3.1 IF Using Frozen Tissue Sections
3.2 IF Using Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue Sections
4 Notes
References
Chapter 18: Complement Detection in Human Tumors by Immunohistochemistry and Immunofluorescence
1 Introduction
2 Materials
2.1 Buffers
2.2 Antibodies
2.3 Common for IHC and Immunofluorescense (IF)
2.4 Specific for IHC
2.5 Specific for IF
3 Methods
3.1 Heating
3.2 Deparaffinization and Rehydration
3.3 Antigen Retrieval
4 Staining
4.1 IHC Staining: C1q, C4d, and C3d (Figs. 1, 2, and 3)
4.2 Classical Double Immunofluorescence Staining: C1q/CD31 (Fig. 4)
4.3 Tyramide Signal Amplification Multiplex Immunofluorescence Staining: C1q/C4d (Fig. 5)
5 Mounting
6 Scan
7 Notes
References
Chapter 19: Analysis of the Ligand Recognition Specificities of Human Ficolins Using Surface Plasmon Resonance
1 Introduction
2 Materials
2.1 SPR Consumables and Buffers
2.2 Immobilized Ligands
2.2.1 Protein Ligands
2.2.2 Sulfated Ligands
2.3 Soluble Protein Analytes
2.3.1 Recombinant Ficolin-1
2.3.2 Recombinant Ficolin-2
2.3.3 Recombinant Ficolin-3
3 Methods
3.1 Immobilization of BSA Glycoconjugates
3.1.1 Immobilization of the BSA Reference Ligand
3.1.2 Immobilization of BSA Glycoconjugates and Acetylated BSA
3.2 Immobilization of Heparin
3.2.1 Immobilization of Streptavidin
3.2.2 Immobilization of Biotinylated Heparin
3.3 Immobilization of the Three Ficolins
3.4 Characterization of the Binding Specificity of Recombinant Human Ficolins
3.5 Kinetic Analysis of Ficolins Binding to Immobilized Ac-BSA
3.5.1 Generation of the Binding Data
3.5.2 Evaluation of the Binding Data
3.6 Influence of the Oligomerization State of Ficolins on Their Binding Properties
3.6.1 Binding Properties of the Isolated FBG Domains of Ficolins
3.6.2 Binding Properties of Recombinant Ficolin-2 Produced in Mammalian and Insect Cells
3.7 Heparin Binding Properties of Ficolins
3.7.1 Binding of the Three Ficolins to Heparin and Kinetic Analysis for Ficolin-2
3.7.2 Competition of Sucrose Octasulfate (SOS) for Ficolin-2 Binding to Heparin
3.8 Binding of Acetylated BSA to Immobilized Ficolins
4 Notes
References
Chapter 20: Methods for Assessment of Interactions of Proteins with Heme: Application for Complement Proteins and Immunoglobul...
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers and Solutions
2.3 Heme
2.4 Proteins (See Note 1)
3 Methods
3.1 UV-Vis Absorbance Spectroscopy
3.2 Biosensor-Based Assay for Measurement Binding Kinetics and Affinity of Heme
4 Notes
References
Chapter 21: Evaluation of Binding Kinetics and Thermodynamics of Antibody-Antigen Interactions and Interactions Involving Comp...
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers and Solutions
2.3 Proteins and Analytes
3 Methods
4 Notes
References
Chapter 22: Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications
1 Introduction
1.1 Overall Structure of Complement C4
1.2 Convertase Assembly and Regulation of C4b
1.3 Sample Preparation for Structural Studies Involving C4 and C4b
2 Materials
2.1 C4 Purification
2.2 C4b Generation
2.3 C4b Deglycosylation for Crystallization and Sample Preparation for X-Ray Crystallography Data Collection
2.4 C4b Deglycosylation and Sample Preparation for SAXS Data Collection
2.5 Sample Preparation for Negative Stain Electron Microscopy and Data Collection
3 Methods
3.1 C4 Purification
3.2 C4b Generation
3.3 C4b Deglycosylation for Crystallization and Sample Preparation for X-Ray Crystallography Data Collection
3.4 C4b Deglycosylation and Sample Preparation for SAXS Data Collection
3.5 Sample Preparation for Negative Stain Electron Microscopy and Data Collection
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2227

Lubka T. Roumenina Editor

The Complement System Innovative Diagnostic and Research Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

The Complement System Innovative Diagnostic and Research Protocols

Edited by

Lubka T. Roumenina Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, Paris, France

Editor Lubka T. Roumenina Centre de Recherche des Cordeliers INSERM, Sorbonne Universite´, USPC Universite´ Paris Descartes, Universite´ Paris Diderot Paris, France

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1015-2 ISBN 978-1-0716-1016-9 (eBook) https://doi.org/10.1007/978-1-0716-1016-9 © Springer Science+Business Media, LLC, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface The complement system has been considered for decades as a simple cell-destroying plasmatic cascade with actions limited in the defense against pathogens and complementing the action of antibodies. Discoveries during the recent years clearly demonstrated that complement actually plays a major role in the host homeostasis, immune surveillance, and pathogenesis of noninfectious diseases. Complement is considered by students and even immunologists as the dark side of immunology not only because of its confusing nomenclature but also because of its complex mode of action. Nevertheless, with the increasing knowledge about the contribution of complement to disease pathogenesis and progression and in the advent of complement therapeutics, this plasmatic cascade came into vogue. To explore it in clinic or in research, we need a large set of robust methods and deep knowledge on its intricate mechanisms in order to analyze it and generate reliable results. With this book, we aim to describe at least part of the methods, which the expert complement laboratories use, and the way that obtained data can be interpreted. Paris, France

Lubka T. Roumenina

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 The Benefits of Complement Measurements for the Clinical Practice . . . . . . . . . . 1 Anne Grunenwald and Lubka T. Roumenina 2 Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Seline A. Zwarthoff, Simone Magnoni, Piet C. Aerts, Kok P. M. van Kessel, and Suzan H. M. Rooijakkers 3 Quantification of Complement Proteins with Special Reference to C1q: Multiplex Versus ELISA Versus Rocket Immunoelectrophoresis Versus Nephelometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Kerstin Sandholm, Barbro Persson, Sliva Abdalla, Camilla Mohlin, Bo Nilsson, and Kristina N. Ekdahl 4 C3dg Quantification by PEG Precipitation and or TRIFMA . . . . . . . . . . . . . . . . . 43 Anne Troldborg and Jens Christian Jensenius 5 Quantification of Porcine Complement Activation Fragment C3a by a Neoepitope-Based Enzyme-Linked Immunosorbent Assay. . . . . . . . . . . . . . . 51 Per H. Nilsson, Kristin Pettersen, Martin Oppermann, Espen W. Skjeflo, Hilde Fure, Dorte Christiansen, and Tom Eirik Mollnes 6 Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Melchior Chabannes, Pauline Bordereau, Paula Vieira Martins, and Marie-Agne`s Dragon-Durey 7 Hemolytic Tests Exploring Factor H Functional Activities . . . . . . . . . . . . . . . . . . . 69 Melchior Chabannes, Shambhuprasad K. Togarsimalemath, and Marie-Agne`s Dragon-Durey 8 Functional Hemolytic Test for Complement Alternative Pathway Convertase Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Marloes A. H. M. Michels, Nicole C. A. J. van de Kar, Elena B. Volokhina, and Bert(L) P. W. J. van den Heuvel 9 Complement C3 Deposition on Endothelial Cells Revealed by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Idris Boudhabhay, Anne Grunenwald, and Lubka T. Roumenina 10 Anti-C1q Autoantibodies: Standard Quantification and Innovative ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Kinga Csorba, Lucia Schirmbeck, Denise Dubler, and Marten Trendelenburg 11 Anti-C1-Inhibitor Autoantibody Detection by ELISA. . . . . . . . . . . . . . . . . . . . . . . 115 Chiara Suffritti, Sonia Caccia, Silvia Berra, Debora Parolin, and Marco Cicardi

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Contents

Anti-Ficolin-2 and Anti-Ficolin-3 Autoantibody Detection by ELISA . . . . . . . . . Chantal Dumestre-Pe´rard and Nicole M. Thielens Detection of Anti-C3b Autoantibodies by ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . Maria Radanova, Lubka T. Roumenina, and Vasil Vasilev Detection of Complement Factor B Autoantibodies by ELISA . . . . . . . . . . . . . . . Miha´ly Jo zsi and Barbara Uzonyi Detection of C3 Nephritic Factor by Hemolytic Assay. . . . . . . . . . . . . . . . . . . . . . . Melchior Chabannes, Ve´ronique Fre´meaux-Bacchi, and Sophie Chauvet Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA . . . . . . . . Jesu´s Garcı´a-Ferna´ndez, Susana Vilches-Arroyo, Leticia Olavarrieta, Julia´n Pe´rez-Pe´rez, and Santiago Rodrı´guez de Cordoba Complement Detection in Mouse Kidneys by Immunofluorescence . . . . . . . . . . . Jennifer Laskowski and Joshua M. Thurman Complement Detection in Human Tumors by Immunohistochemistry and Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marie V. Daugan, Margot Revel, Laetitia Lacroix, Catherine Saute`s-Fridman, Wolf H. Fridman, and Lubka T. Roumenina Analysis of the Ligand Recognition Specificities of Human Ficolins Using Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicole M. Thielens, Evelyne Gout, Monique Lacroix, Jean-Baptiste Reiser, and Christine Gaboriaud Methods for Assessment of Interactions of Proteins with Heme: Application for Complement Proteins and Immunoglobulins . . . . . . . . . . . . . . . . Margot Revel and Jordan D. Dimitrov Evaluation of Binding Kinetics and Thermodynamics of Antibody–Antigen Interactions and Interactions Involving Complement Proteins. . . . . . . . . . . . . . . . Sofia Rossini and Jordan D. Dimitrov Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alessandra Zarantonello, Sofia Mortensen, Nick S. Laursen, and Gregers R. Andersen

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors SLIVA ABDALLA • Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden PIET C. AERTS • Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands GREGERS R. ANDERSEN • Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark SILVIA BERRA • Department of Biomedical and Clinical Sciences “L. Sacco”, University of Milan, Milan, Italy PAULINE BORDEREAU • Service d’Immunologie Biologique, Hoˆpital Europe´en Georges Pompidou, Assistance Publique-Hoˆpitaux de Paris, Paris, France IDRIS BOUDHABHAY • Centre de Recherche des Cordeliers, INSERM, Sorbonne Universite´, USPC, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France SONIA CACCIA • Department of Biomedical and Clinical Sciences “L. Sacco”, University of Milan, Milan, Italy MELCHIOR CHABANNES • INSERM UMRS 1138, “Inflammation, Complement and Cancer” Team, Centre de Recherche des Cordeliers, Sorbonne Universite´, Universite´ de Paris, Paris, France SOPHIE CHAUVET • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France; Service de Ne´phrologie, Hoˆpital europe´en Georges Pompidou, APHP, Paris, France DORTE CHRISTIANSEN • Research Laboratory, Nordland Hospital, Bodø, Norway MARCO CICARDI • ASST-Fatebenefratelli-Sacco, Milan, Italy; Department of Biomedical and Clinical Sciences “L. Sacco”, University of Milan, Milan, Italy KINGA CSORBA • All Clinical Immunology Laboratory, Division of Internal Medicine and Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland MARIE V. DAUGAN • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France SANTIAGO RODRI´GUEZ DE CO´RDOBA • Centro de Investigaciones Biologicas and Ciber de Enfermedades Raras, Madrid, Spain JORDAN D. DIMITROV • Centre de Recherche des Cordeliers, INSERM, Sorbonne Universite´, USPC, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France MARIE-AGNE`S DRAGON-DUREY • INSERM, UMRS 1138, “Inflammation, Complement and Cancer” Team, Centre de Recherche des Cordeliers, Sorbonne Universite´, Universite´ de Paris, Paris, France; Service d’Immunologie Biologique, Hoˆpital Europe´en Georges Pompidou, Assistance Publique-Hoˆpitaux de Paris, Paris, France DENISE DUBLER • All Clinical Immunology Laboratory, Division of Internal Medicine and Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland CHANTAL DUMESTRE-PE´RARD • Laboratoire d’Immunologie, Poˆle de Biologie, CHU Grenoble Alpes, Grenoble Cedex 9, France; BNI TIMC-IMAG, UMR5525, CNRS-Universite´ Grenoble Alpes, Grenoble Cedex 9, France

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Contributors

KRISTINA N. EKDAHL • Centre of Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden; Department of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory C5:3, Uppsala University, Uppsala, Sweden VE´RONIQUE FRE´MEAUX-BACCHI • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France; Service d’Immunologie, Hoˆpital europe´en Georges Pompidou, APHP, Paris, France WOLF H. FRIDMAN • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France HILDE FURE • Research Laboratory, Nordland Hospital, Bodø, Norway CHRISTINE GABORIAUD • Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France JESU´S GARCI´A-FERNA´NDEZ • Centro de Investigaciones Biologicas and Ciber de Enfermedades Raras, Madrid, Spain EVELYNE GOUT • Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France ANNE GRUNENWALD • Centre de Recherche des Cordeliers, INSERM, Sorbonne Universite´, USPC, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France JENS CHRISTIAN JENSENIUS • Department of Biomedicine, Aarhus University, Aarhus, Denmark MIHA´LY JO´ZSI • Department of Immunology, ELTE Eo¨tvo¨s Lora´nd University, Budapest, Hungary LAETITIA LACROIX • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France MONIQUE LACROIX • Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France JENNIFER LASKOWSKI • Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA NICK S. LAURSEN • Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark SIMONE MAGNONI • Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands PAULA VIEIRA MARTINS • Service d’Immunologie Biologique, Hoˆpital Europe´en Georges Pompidou, Assistance Publique-Hoˆpitaux de Paris, Paris, France MARLOES A. H. M. MICHELS • Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, The Netherlands CAMILLA MOHLIN • Centre of Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden TOM EIRIK MOLLNES • Department of Immunology, University of Oslo and Oslo University Hospital Rikshospitalet, Oslo, Norway; Faculty of Health Sciences, K.G Jebsen TREC, UiT—The Arctic University of Norway, Tromsø, Norway; Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway SOFIA MORTENSEN • Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark BO NILSSON • Department of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory C5:3, Uppsala University, Uppsala, Sweden

Contributors

xi

PER H. NILSSON • Department of Immunology, University of Oslo and Oslo University Hospital Rikshospitalet, Oslo, Norway; Linnaeus Centre for Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden LETICIA OLAVARRIETA • Secugen S.L., Madrid, Spain MARTIN OPPERMANN • Institute for Cellular and Molecular Immunology, University Medical Center, Georg-August-University Go¨ttingen, Go¨ttingen, Germany DEBORA PAROLIN • Department of Biomedical and Clinical Sciences “L. Sacco”, University of Milan, Milan, Italy JULIA´N PE´REZ-PE´REZ • Secugen S.L., Madrid, Spain BARBRO PERSSON • Department of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory C5:3, Uppsala University, Uppsala, Sweden; Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden KRISTIN PETTERSEN • Research Laboratory, Nordland Hospital, Bodø, Norway MARIA RADANOVA • Department of Biochemistry, Molecular Medicine and Nutrigenomics, Medical University of Varna, Varna, Bulgaria JEAN-BAPTISTE REISER • Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France MARGOT REVEL • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France SUZAN H. M. ROOIJAKKERS • Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands SOFIA ROSSINI • Centre de Recherche des Cordeliers, INSERM, Sorbonne Universite´, USPC, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France LUBKA T. ROUMENINA • Centre de Recherche des Cordeliers, INSERM, Sorbonne Universite´, USPC, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France KERSTIN SANDHOLM • Centre of Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden CATHERINE SAUTE`S-FRIDMAN • INSERM, UMR_S 1138, Inflammation, Complement and Cancer Team, Centre de Recherche des Cordeliers, Sorbonne Universite´s, Universite´ de Paris, Paris, France LUCIA SCHIRMBECK • All Clinical Immunology Laboratory, Division of Internal Medicine and Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland ESPEN W. SKJEFLO • Research Laboratory, Nordland Hospital, Bodø, Norway; Faculty of Health Sciences, K.G Jebsen TREC, UiT—The Arctic University of Norway, Tromsø, Norway CHIARA SUFFRITTI • ASST-Fatebenefratelli-Sacco, Milan, Italy NICOLE M. THIELENS • Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France JOSHUA M. THURMAN • Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA SHAMBHUPRASAD K. TOGARSIMALEMATH • INSERM, UMRS 1138, “Inflammation, Complement and Cancer” Team, Centre de Recherche des Cordeliers, Sorbonne Universite´, Universite´ de Paris, Paris, France MARTEN TRENDELENBURG • All Clinical Immunology Laboratory, Division of Internal Medicine and Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland ANNE TROLDBORG • Department of Biomedicine, Aarhus University, Aarhus, Denmark; Department of Rheumatology, Aarhus University Hospital, Aarhus, Denmark

xii

Contributors

BARBARA UZONYI • Department of Immunology, ELTE Eo¨tvo¨s Lora´nd University, Budapest, Hungary NICOLE C. A. J. VAN DE KAR • Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, The Netherlands BERT(L) P. W. J. VAN DEN HEUVEL • Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, The Netherlands; Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands; Department of Pediatrics/ Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium; Department of Development and Regeneration, University Hospitals Leuven, Leuven, Belgium KOK P. M. VAN KESSEL • Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands VASIL VASILEV • Clinic of Nephrology, University Hospital—“Tzaritza Yoanna—ISUL”, Medical University of Sofia, Sofia, Bulgaria SUSANA VILCHES-ARROYO • Secugen S.L., Madrid, Spain ELENA B. VOLOKHINA • Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, The Netherlands; Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands ALESSANDRA ZARANTONELLO • Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark SELINE A. ZWARTHOFF • Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

Chapter 1 The Benefits of Complement Measurements for the Clinical Practice Anne Grunenwald and Lubka T. Roumenina Abstract The complement cascade is an evolutionary ancient innate immune defense system, playing a major role in the defense against infections. Its function in maintaining host homeostasis on activated cells has been emphasized by the crucial role of its overactivation in ever growing number of diseases, such as atypical hemolytic uremic syndrome (aHUS), autoimmune diseases as systemic lupus erythematosus (SLE), C3 glomerulopathies (C3GN), age-related macular degeneration (AMD), graft rejection, Alzheimer disease, and cancer, to name just a few. The last decade of research on complement has extended its implication in many pathological processes, offering new insights to potential therapeutic targets and asserting the necessity of reliable, sensitive, specific, accurate, and reproducible biomarkers to decipher complement role in pathology. We need to evaluate accurately which pathway or role should be targeted pharmacologically, and optimize treatment efficacy versus toxicity. This chapter is an introduction to the role of complement in human diseases and the use of complement-related biomarkers in the clinical practice. It is a part of a book intending to give reliable and standardized methods to evaluate complement according to nowadays needs and knowledge. Key words Complement system, Complement biomarkers, Complement activation, Complement mediated diseases, Complement therapeutics

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Complement system in Physiology Complement is one of the most conserved and ancient parts of the host defense against infections throughout evolution. Its best appreciated role is the defense against pathogens by opsonization or lysis and by inducing an inflammatory response from immune cells [1]. Complement is a family of around 50 proteins susceptible to activation through various triggers in a tightly regulated way. Most of the complement proteins are produced by the liver [2]. Complement progresses in a cascade manner until C5b-9 membrane attack complex (MAC) formation, which is the most well-known and traditional view [1]. Three different pathways can lead to this terminal complement pathway activation, all leading to C3 cleavage

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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in C3a and C3b by C3 convertases, keystone of complement activation. Assembly of the classical C3 convertase (C4b2a) is the result of activation of the classical pathway (by binding of C1q to immune complexes) and lectin pathway (by recognition of glucidic residues such as mannose—by mannose binding lectin, fucose—by Collectin 11, or N-acetylglucosamine—by ficolins, all recruiting Mannanbinding lectin associated serine proteases, MASP1 and MASP2). The alternative pathway is constitutively activated at low level by spontaneous hydrolysis, in solution or in contact with an interface, of C3 to C3(H2O). C3(H2O) interacts with factors B and D (FB, FD) to form a transient C3 convertase C3(H2O)Bb, allowing cleavage of C3 to C3a and C3b. C3b is able to bind covalently to cell surface. In absence of complement regulators, C3b can recruit FB and FD to form an alternative C3 convertase (C3bBb), allowing for an amplification loop for C3b generation. The alternative pathway C3 convertase can be stabilized by properdin [3]. C3b can also bind a classical or alternative C3 convertase to form a classical (C4b2a3b) or an alternative (C3bBb3b) C5 convertase, able to cleave C5 in C5a and C5b which will recruit C6, C7, C8, and C9 to form the MAC. MAC is able to lyse bacteria or erythrocytes and to activate the other host cells [1]. Tight regulation is exerted by membrane regulators, which can dissociate the classical (C4 binding protein, C4bp; Complement Receptor 1 (CR1, CD35), Decay accelerating Factor (DAF, CD55)) or the alternative (factor H (FH), DAF, and CR1) C3 convertase. Moreover, degradation of C4b and C3b by factor I and its cofactors FH, CR1, Membrane Cofactor Protein (MCP, CD46) result to generation of fragments unable to assemble new C3 convertases: C4d and iC3b. Complement anaphylatoxins C3a and C5a induce inflammation and activate immune and nonimmune cells (endothelial, epithelial, etc.) through their specific receptors C3aR and C5aR1, C5aR2, modulating adaptive immune response and tissue inflammation. Apart from this “canonical” pattern of activation through the cascade, evidence grows that complement can be activated “noncanonically.” One example is the autocrine way when complement components are released by cells (in fact, almost all cell types can produce at least part of the complement proteins [4]) which become a target of their own complement proteins . Another example is the intracellular activation of the complement proteins [5]. This activation occurs by a convertase-independent pathway, likely due to cleavage, dependent on cathepsin L [6]. Convertaseindependent cleavage was reported also for renin [7], thrombin [8], and plasmin [9], able to cleave C3 and C5 [10]. Nevertheless, further studies are needed to confirm this convertase-independent cleavage. Furthermore, in lectin pathway, MASP2 can directly activate C3 and bypass C2 and C4 [11]. In vivo mice studies showed that local complement release had several crucial functions such as restoring impaired humoral response to tumor [12], favoring T cell

Complement in the Clinical Practice

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activation and orientation by antigen presenting cells (APC) [13, 14] and accelerating cellular rejection during kidney transplantation [15]. Intracellular complement activation, which is a recent and very promising new field into complement deep comprehension, is described as essential to T cell specialization [5, 16]. In CD4 + T cells, intracellular complement activation (via C3a-C3aR, C5a-C5aR1) is essential for TLR-dependent survival [17], for transcription, for metabolism and for Th1 differentiation, through IFNγ production depending of CD46 and NLRP3 inflammasome [18]. Intracellular complement components are also essential for metabolism, transcription, and survival in other immune cells such as B cells and CD8+ T cells [18–20]. However, these intracellular functions of complement are also subject to criticism as its subcellular localization is still unclear (organelles, cytoplasm, or nucleus) and as its origin is not proven (uptake from extracellular medium, retrotranslocation of partially processed C3 to cytosol, etc.). However, these debates suggest wide new fields waiting to be unraveled in complement physiological functions.

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Role of Complement in Pathology First role known for complement is defense against pathogens by phagocytosis or by direct lysis, consequence of MAC formation. The C5b-9 complex is a 10 nm wide pore in the target membrane, induced by activation of each of the three pathways depending on pathogen-associated molecular patterns [21]. For many pathogens, resistance mechanisms to complement lysis and other defense pathways make complement system redundant. However, complete deficits in complement proteins, rare and mostly recessive diseases [22, 23], are all associated with an increased risk of encapsulated bacterial infections such as Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus sp. with clinical display of meningitides, bacteremia, pneumonia, and upper respiratory tract infections, depending on the deficient protein [24]. As Neisseria species are very sensitive to MAC formation, their incidence is increased in patients deficient in terminal complement components (C5 to C9) as well as in properdin [22]. In early complement proteins deficiencies, bacterial infections will be associated with viral and protozoan infection when lectin pathway is involved and increased incidence of autoimmune disease like SLE when classical pathway is involved. Several pathologies—thrombotic, inflammatory, autoimmune, and age-related diseases—are associated with abnormal complement activation. The major discoveries of last two decades in atypical hemolytic uremic syndrome (aHUS), evidenced complement pathway dysregulation as the keystone in the pathophysiology of this renal

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thrombotic microangiopathy. Activating (in genes coding for protein of the cascade) or inactivating (in regulators genes) genetic mutations and acquired autoantibodies lead to proinflammatory activation of endothelium by complement cascade, leading to microthrombi formation with consumption of platelets, especially in the kidney, responsible for a mechanical hemolytic anemia. Clinically, it is responsible for an acute kidney injury. However, aHUS has incomplete penetrance in genetic variants and 40% of etiologies remain unknown. Activation of complement has been also associated with other etiologies of thrombotic microangiopathies such as typical HUS, HELLP syndrome, and some infection-related thrombotic microangiopathies [25]. C3 glomerulopathies are another example of rare diseases, associated with complement dysregulation. This heterogeneous group of diseases is characterized by predominant glomerular C3 deposits in mesangium and along glomerular basement. Clinically, this condition is associated with variable ranges of hematuria, proteinuria, hypertension, and kidney injury, leading potentially to kidney failure. C3 glomerulopathies encompass dense deposit diseases with osmiophilic “sausage-shaped” deposits in electron microscopy, C3 glomerulonephritis (C3GN) but also C3 predominant postinfectious glomerulonephritis (PIGN) [26]. Complement over activation is frequently secondary to a convertase stabilizing antibody, a nephritic factor (C3Nef, C5Nef, C4Nef [27]). In rare cases anti-C3b and anti-FB antibodies have been reported in C3G [28]. Anti-FB are more common in acute pediatric PIGN [29], while anti-C3b—in lupus nephritis [30]. Ig-associated membranoproliferative glomerulonephritis, which was historically opposed to C3 glomerulopathies, is also associated with complement over activation, suggesting similar pathogenic mechanisms [28, 31]. Besides these acquired factors, about 25% of patients with C3G carry rare or unique variants in complement-related genes [26, 32, 33] with gene-specific clusters of mutations (amino-terminal portion of complement factor H CFH for example) or rearrangement in CFH locus in familial C3G. Complement activation has been involved in an increasing number of rheumatic diseases with, in common, deposition in tissues of activation fragments, high level of activated complement products in circulation and decreased residual function. Among them a key example is the systemic lupus erythematosus (SLE) in which congenital deficiencies of the early classical pathway proteins (C1q, C2, and C4) are among the strongest genetic risk factors [34]. Alteration in phagocytosis of apoptotic cells, especially in the absence of C1q, could stimulate an inappropriate immune response to autoantigens [35–37]. More recently it has been shown that C1q modulates the metabolism of CD8 T-cells, explaining thus the link between C1q deficiency and SLE [19]. In SLE, inflammation is associated with enhanced autoantibodies, among

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them anti-C1q antibodies [38] and anti-C3b [30], and immune complexes capable of complement classical pathway activation and tissue deposition, like in lupus glomerulonephritis [39]. Glomerular deposits are often associated with consumption of circulating complement (with decreased C4 and C3 levels). This underlines the double-edged sword of complement in SLE [36]. In rheumatoid arthritis, complement activation fragments are increased in synovial fluid [40] triggered by immune complexes [41], dead cells, extracellular DNA, and so on [37]. However, in animal models complement is unexpectedly mostly dependent of alternative pathway [42]. Active stage of anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis is associated with increased circulating complement activation fragments [43] and renal deposition [44], attributed to neutrophils release of activating factors of alternative pathway like properdin and extracellular traps, involved in complement activation [45]. In cancerology, during the past decade, the role of complement started to emerge. Complement proteins are synthesized in the tumor microenvironment (TME) [10, 46]. Complement is likely activated by the classical pathway [47] but also in a noncanonical way, by plasmin [9]. Its activation fragments can exert, depending of tumor type, pro or anti tumoral effects by modulating tumor microenvironment [10]: In mouse models, C3a and C5a play the role of chemokines toward immune cells [48–50], influence cytokine production, and upregulate growth factor expression to promote angiogenesis, and C1q was recently involved in cancer neoangiogenesis via a noncanonical, cascade independent mechanism [47, 51]. In murine models, complement can also affect survival and proliferation of tumor cells in an autocrine manner. In human cancers, complement expression varies greatly among tumor types depicting different patterns of tumor in which complement can be protective or aggressive [10]. Complement is also involved in aging related immunodegenerative phenotypes such as neuroinflammatory diseases and neurodegenerative diseases [52, 53]. In animal models of development, C1q and interaction between CR3 on microglial cells and activation fragments of C3 on synapses are involved in development related synapse refinement [54, 55]. In Alzheimer’s disease (AD), characterized by neuroinflammation (activation of astrocytes and microglia) and amyloid-β (Aβ) plaque and neurofibrillary tangles, increased deposition of complement has been evidenced [56]. C1q and C3 have been involved in phagocytic clearance of amyloid fibrils and C5a and MAC have been related to demyelination, neuronal loss, and inflammation [57, 58]. Single Nucleotide Polymorphisms (SNP) associated with risk of AD were identified in CR1 [59]. However mouse model of AD suggest a dual role for complement as C1q deletion was associate with attenuated neuropathology but increased C3 deposition and plaque [60] like in C3KO

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mice [61]. In amyotrophic lateral sclerosis (ALS) caused by progressive loss of upper and lower motoneurons, associated with neuroinflammation (astrocytes and microglia activation and T cell infiltration), deposition of complement activation fragments [62] are increased together with plasmatic complement biomarkers [63] which are also correlated with severity. Complement C1q, C4, or C3 deletion [64, 65] in a murine model of amyotrophic lateral sclerosis did not provide protection but inhibition of C5aR reduced weight loss, motor deficit, and mortality [66, 67]. Complement deposition has also been evidenced in Huntington’s disease [68] and Parkinson’s disease [69]. Myasthenia gravis is characterized by severe muscle weakness consequence of autoantibody-mediated destruction of neuromuscular junction mediated in around 80% by anti-acetylcholine receptor (AChR) Antibody which could recognize different epitopes and either block synaptic transmission, accelerate normal AChR internalization and turnover, or activate the complement cascade [70, 71] Neuromuscular junction damage was indeed MAC dependent in a mouse model of myasthenia gravis and complement deposition was evidenced at the endplate in pateints [72]. Age-related macular degeneration (AMD) is the major cause of blindness among elderly patients, has been for long associated with FH polymorphisms [73]. Mutations in CFI, C3 and C9 are also associated with high risk of advanced AMD [74]. In case of the SNP in FH (Y402H variant), complement activation by photoinduced oxidized lipids and malondialdehyde is not controlled, leading to retinal epithelial cells damage and macrophages activation [75, 76] responsible for irreversible loss of photoreceptors [77]. Consistent with this, genetic deletion of FH competitors CFHR1 and CFHR3 is protective for AMD [78]. Recently FHR-4 demonstrated to play a prominent role in with elevated systemic FHR-4 levels in AMD with accumulation of FHR-4 in the choriocapillaris, Bruch’s membrane and drusen, competing with FH/FHL-1 for C3b binding [79]. Paroxysmal nocturnal hemoglobinuria (PNH), displaying hemolytic anemia, bone marrow failure, and thrombosis, is a clonal hematopoietic stem cell disorder resulting from a somatic mutation in PIGA gene which is necessary for biosynthesis of GPI, a glycolipid fragment anchoring several proteins to the cell surface. Among them, complement regulators CD55 and CD59, are missing at hematopoietic cells surface, permitting alternative pathway to activate spontaneously, enhanced by inflammatory conditions [80]. This activation is partly responsible for thrombotic events [81]. Improvement of patients with anti-C5 therapies, revealed a part of C3 mediated extravascular hemolysis mediated by C3dg [82–84].

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Sickle cell disease (SCD), and its homozygous form sickle cell anemia (SCA), is a highly prevalent genetic disease characterized by predominance of mutated Hemoglobin S responsible for vasoocclusive events secondary to red blood cell deformation under particular conditions leading to organ injuries. It has early been associated with enhanced complement activation product C3c deposition on RBCs [85–88], enhanced soluble sC5b-9 in fluid phase [88] and deposition of complement activation products in patients tissues [87, 89]. In an in vivo model of SCD, complement inhibition by a C5 inhibitor permitted quicker reversion of vasoocclusion [90] and prevented liver dysfunction [91]. Moreover, complement system is a key participant in the pathophysiology of posttransfusion hemolysis, including in SCA [92]. Complement can trigger the hemolytic reaction, amplify the inflammatory response and increase tissue damage.

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Complement as a Drug Target The story of targeted complement treatment, apart from complement supplementation or replacement by plasmatic exchanges, started with C1 inhibitor purification in the 80s [93] to supply patients with hereditary angioedema. But it is not before the beginning of 2000’s that human complement inhibition revolutionized the treatment of pathologies with excessive complement activation, with the testing of the anti-C5 inhibitory antibody eculizumab (Soliris, Alexion) in a pilot study in PNH [94]. Its efficacy and safety have not only been evidenced in PNH [95–97] but transformed renal prognostic in atypical HUS [98–100]. In HELLP syndrome, in infection-associated HUS, and particularly in transplantation associated HUS, eculizumab showed encouraging results but further investigations are needed [101–103]. However, if eculizumab dramatically alters natural course of PNH and aHUS, its efficacy is uncomplete in PNH (1/3 with complete response characterized by hemoglobin normalization [82]) and aHUS (around 70% efficacy [104]). It can be caused by intrinsic resistance to eculizumab [105], pharmacokinetic breakthrough (associated with low plasma level of eculizumab) [106], and pharmacodynamics breakthrough (role of C3 mediated extravascular hemolysis [82] and variation of C5 convertase affinity to C5 substrate [107]). Possibility of treatment interruption in acute diseases like aHUS has been evidenced with among 30% of relapses [108–110]. To challenge this limitations while competing eculizumab economic burden and improving galenic administration, many complement inhibitors have been designed and are under clinical development [111] (Table 1). These new molecules target not only C5 and terminal activation pathway but also proximal

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Table 1 Nonexhaustive complement inhibitors in current development for complement-mediated indications Complement therapeutic

Mechanism of action Interest in

State of development

Target

Entity

C5

mAb

Inhibition of HUS, PNH, Eculizumab, C5 activation myasthenia Alexion gravis, RNO, Pharmaceuticals transplantation, C3G, Guillain Barre´ syndrome, AMD

mAb

Ravulizumab (ALXN1210), Alexion

Same epitope as HUS, PNH, eculizumab myasthenia gravis, RNO, ALS

In clinic Phase III

mAb

ABP959, Amgen

Same epitope as PNH, aHUS eculizumab

Phase III

mAb

SB12, Samsung bioepis

Same epitope as PNH eculizumab

Phase III

mAb

SKY59, Hoffman—La Roche

Different PNH epitope from eculizumab

Phase I/II

mAb

Tesidolumab/ LFG316/ Novartis

Different PNH epitope from eculizumab

Phase II

mAb

Pozelimab (REGN3918), Regeneron

PNH, Inhibition of gastrointestinal C5 activation disorders (wild-type and R885H variant of human C5)

Phase III Phase II/III

Protein

Nomocopan/ coversin, Akari

C5 inhibitor which also inhibits leukotriene B4 (LTB4)

HUS, PNH, bullous pemphigoid, TMA, uveitis

Phase III Phase II Phase I

Oligonucleotide Cemdisiran/ ALN-CC5, Alnylam

RNAi of C5

HUS, PNH, IgAN

Phase II

Oligonucleotide Zimura/ avacincaptad pegol, Ophthotech Corp

C5 cleavage inhibitor

AMD

Phase II

In clinic Phase III Phase II

(continued)

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Table 1 (continued)

Entity

Complement therapeutic

Macrocyclic peptid

Zilucoplan, RA C5 cleavage pharmaceuticals inhibitor

Myasthenia gravis, IM myopathy, PNH, HUS, lupus nephritis

Phase III Phase II Phase I

C5aR

Small molecule

Inhibition of Avacopan/ C5aR1 CCX168 signaling ChemoCentryx

ANCA vasculitis, C3G, aHUS, IgAN, HS

Phase III Phase II

C5aR1

mAb

IPH5401, innate pharma

Inhibition of C5aR1

Solid tumors

Phase I

C5a

mAb

IFX-1, InflaRx

Inhibition of C5a activity

Sepsis, HS, GPA, PG

Phase II

C3

Peptide

AMY-101, Amyndas

Peptidic analog Periodontal disease, Phase II Phase I PNH, C3G, of AMD, compstatin, transplantation, inhibits C3 renal failure cleavage

Peptide

Pegcetacoplan/ APL-2, Apellis

Pegylated derivative of compstatin, inhibits C3 cleavage

Target

Mechanism of action Interest in

State of development

PNH, AMD, AIHA, C3G, lupus nephritis, AMD

Phase III Phase II Phase I/II

Transplantation, kidney ischemia reperfusion

Phase III Phase II

Convertase Protein

CR1 analog Mirococept, Inlammazyme pharmaceuticals

FD

Small molecule

Danicopan/ ACH-4471, Achillion

Inhibits FD and PNH, C3G thus cleavage of complement factor B

FB

Molecule

LNP023, Novartis

Binds to FB, preventing C3bBb formation

C3G, PNH, IgAN

Phase II

Inhibition of hepatic FB expression

AMD

Phase II

Oligonucleotide IONIS-LB-LRx, Roche Properdin

mAb

CLG561, Alcon/ Inhibition AP AMD Novartis amplification

C1r, C1s Human protein Berinert/CSL et MASP Behring Cinryze, Shire Cetor, Sanquin

Human C1 esterase inhibitor

C1 inhibitor deficiency, transplantation, kidney failure

Phase II

Phase I In clinic Phase II Phase I/II (continued)

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Anne Grunenwald and Lubka T. Roumenina

Table 1 (continued)

Entity

Complement therapeutic

Mechanism of action Interest in

State of development

Recombinant protein

Conestat alfa, pharming

Recombinant C1 esterase inhibitor

C1 inhibitor deficiency, contrast-induced nephropathy, preeclampsia, transplantation

Phase II Phase I

C1s

mAb

Sutimlimab/ BIVV009, Bioverativ

C1s inhibitor

AIHA ITP

Phase III Phase I

DR5

HexaBody (ab)

GEN1029, GenMab

Enhancement of CDC against DR5 + tumors by agonist activity

Cancer

Phase I/II

MASP2

mAb

Narsoplimab (OMS721), Omeros

MASP2 inhibitor

MAT, aHUS, IgA nephropathy, C3G

Phase II/III Phase III Phase II

Target

aHUS atypical hemolytic uremic syndrome, AIHA autoimmune hemolytic anemia, AMD age-related macular degeneration, C1-INH C1 esterase inhibitor, C3G C3 glomerulopathy, CDC complement-dependent cytotoxicity, DR5 death receptor 5, Fab antigen-binding fragment, FB Factor B, GPA granulomatosis with polyangiitis, HS Hidradenitis suppurativa, HUS hemolytic uremic syndrome, IgAN IgA nephropathy, IM myopathy Immune-mediated myopathy, ITP Idiopathic thrombocytopenic purpura, I/R ischemia–reperfusion, mAb monoclonal antibody, MASP mannosebinding lectin-associated serine proteases, PG Pyoderma gangrenosum, PNH paroxysmal nocturnal hemoglobinuria, RNO Recurrent neuromyelitis optica, SIRS systemic inflammatory response syndrome, TMA thrombotic microangiopathy

complement pathway with C3, FB, FD, and even initiators of complement activation (MASP-2, C1s, properdin) and complement receptors. Complement inhibition is nowadays a field in huge expansion in many pathologic conditions: in renal transplantation [112] in treatment [113, 114] of antibody mediated rejection and prevention [115] in sensitized patients but also in AMD [116], in central nervous system diseases [53, 117] and in trauma or sepsis [118] with conflicting results. It is also a challenge in autoimmune diseases [119], where terminal complement inhibition improved severe lupus, notably with TMA [120, 121] and myasthenia gravidis [122] and might improve catastrophic antiphospholipid syndrome (CAPS) [123, 124]. In particular, C5aR1 inhibitor CCX168 has promising effect in ANCA vasculitis, enabling reduce exposition to corticoids [125]. Finally in cancer, phase I/II trial are ongoing to test the effect of C5aR inhibition in association with checkpoint inhibitors to increase tumor-elimination efficacy [126].

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Host Cell DAF

CD46

CR1

-

-

iC3b

FH -

ALTERNATIVE PATHWAY C3b(H2O)Bb

Ba

B

C3b Bb

C3b

FI

Alternative C3 convertase

C3

LECTIN PATHWAY MBL, Collectins MASPs

C4a C4

Alternative

C3a C3b

C3bBb3b C5 convertase C5b

C5

C5a Classical C3 convertase

C4b2a3b Classical

FI

C5 convertase

C4b2a C1q C1r C1s

C5b-9

Pathogen

C2 C2b

-

-

CLASSICAL PATHWAY DAF

C4bp

iC3b

CR1

CD46

-

Fig. 1 Schematic representation of complement activation pathways

The use of complement inhibitors (as well as complement deficiency [24]) compromise encapsulated antibacterial immunity and must be associated with antipneumococcal, antimeningococcal and anti-Hemophilus influenzae vaccines, optimally associated with antibioprophylaxis. Data regarding length of antibioprophylaxis after withdrawal of complement inhibitors or its efficacy are missing [127].

4

Challenges for Complement Monitoring Complement status of a patient should be monitored to assess diagnosis in inherited or acquired complement deficiency, to investigate the etiology of diseases with complement overactivation, and to monitor effects on complement regulatory drugs. The diseases for which complement testing is most frequently indicated are the following: in atypical HUS, C3 glomerulopathy, poststreptococcal glomerulonephritis, to evaluate severity and potential sensitivity to complement inhibition in SLE and in antibody-mediated rejection. In case of increased susceptibility and severity to bacterial infections, meningococcal disease, and hereditary angioedema, inherited or acquired complement deficiency should be investigated right away. However, in autoimmune diseases and aHUS

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complement activation should be characterized before genetic analyses. Despite new improvements in complement inhibition, because of the lack of a rapid confirmatory diagnostic assays and of accessible, reliable, and reproductive follow-up markers, treatment might be suboptimal (delayed, not administered, not accurate posology). Indeed, accurate diagnosis and evaluation rely on robust and reliable measurement of complement function, individual complement proteins, and activation products [128]. If activation of complement is routinely assessed by immunochemical measurement of C3, C4, and factor B levels and determination of total hemolytic complement activity (CH50), this approach has several limitations. First, there are a wide range of normal concentrations of some complement components, and concentrations vary in several physiological conditions like pregnancy [129]. Furthermore, small variation in measured circulating complement proteins concentration reflects poorly local complement activation and are of limited prognostic significance as complement can stay in normal range while being highly activated at local level. Then, differences in sampling collection (serum, citrated, or EDTA plasma) are also responsible for concentrations variability as EDTAplasma sampling avoids further complement activation by Ca2+ and Mg2+ chelation but is not applicable in certain tests for complement function analysis. On the contrary, serum contains functionally active complement, but is unstable (can autoactivate) and does not have the same stability under freezing [130]. Variability in conservation processes, and the numerous techniques used around the world are other limitations for sensitive and specific diagnosis and robustness in publications. Concerning activation products (complement activation fragments or protein–protein complexes), they can be quantified by monoclonal antibodies recognizing epitopes hidden in the native protein, called neoepitopes, with a risk of cross-reactivity with the native protein. Moreover, eculizumab-C5 complexes express a C5a neoepitope in vivo, which has consequences for interpretation of patient complement analyses [131]. To evaluate the formation of C5b-9 complex, an enzyme immunoassay can be performed with a capture antibody against a neoepitope of C9 and the sC5b-9 will be then quantified with another antibody recognizing another part of the complex. Complement function is traditionally assessed by hemolytic assays like CH50 or AH50 enabling to measure total hemolytic complement activity [132] but it is also possible to avoid individual variability in erythrocytes by using liposomes [133] or determine which pathway is implicated by microtiter plate assays [134]. The need for more sensitive and specific assays, particularly in the era of effervescence of complement inhibitors, has influenced the emergence of new assays for complement function with, for example, the modified Ham test [135] and study of deposition of complement on endothelial cells [136]. However, these assays are very sensitive

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to autoactivation because they can only be performed in serum (or eventually with citrated plasma with addition of normal serum [137]). Acquired antibodies targeting complements have historically be associated with C3Nef in DDD, discovered in the 1970s, and characterized as an alternative C3 convertase stabilizator [138, 139]. Since then, other nephritic factors stabilizing C5 alternative convertase (C5NeF) [140] and C3/C5 convertase of the classical and lectin pathways (C4NeF) [141–143] have been reported. They have in common the capacity of recognizing neoepitopes of assembled convertase and increasing its half-life [27]. Identification of these nephritic factors can be performed by analyses of breakdown products or binding assays which share the step of in vitro formation of convertase with the technical difficulties of a transient and unstable conformational epitope. Moreover, hemolytic assays, another technique of nephritic factor detection, give rise to the issue of reproducibility as regards red blood cell isolation. Besides nephritic factors, other antibodies have been discovered in aHUS (targeting FH [144, 145]) and in DDD and C3GN, targeting factor B and C3b [28, 146]. To go further into etiologic diagnosis, targeting genetic polymorphisms and rare variants/mutations is of interest in many pathologies [23] associated with impairment of immunity against microorganisms [147, 148] or excessive complement activation such as aHUS [149, 150], AMD [151], and C3 glomerulonephritis [152, 153]. If in many cases, the presence of one or more polymorphisms, increasing susceptibility to complement mediated injuries, is not essential for the diagnosis, in some pathologies, and aHUS is the most striking example of it, identification of a mutation and a polymorphism/at risk haplotype is a key information for etiologic diagnosis, prognosis [150], and treatment. Indeed, treatment is often used as therapeutic test because genetic analysis is delayed compared to disease onset. However, response to eculizumab therapy [110] and relapse after transplantation are dependent on genetic etiology [154]. However, genetic analysis is not spared from several limitations. Firstly, the indications: systematic screening of all patients with biological indications for thrombotic microangiopathy is poorly cost-effective without solid indications for atypical HUS. Secondly, the multiplicity: many mutations have been described in almost all of complement genes in disease cohorts. The position of mutations is the same in C3 glomerulopathies and aHUS for Factor H [153, 155]. Genetic abnormalities occur also due to gene conversion, like for factor H which has five factor H-related proteins CFHR1, CFHR2, CFHR3, CFHR4, and CFHR5. The rearrangements in this cluster occur often in patients, but they are undetectable by Sanger sequencing and nextgeneration DNA sequencing (NGS) analysis approaches [156– 160]. Multiplex ligation-dependent probe amplification (MLPA)

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is needed to detect them. Also, functional assays are required to assess the responsibility of a genetic abnormality or autoantibodies in atypical HUS and C3G. Deletion of CFHR genes can be responsible for the development of antibodies targeting FH in aHUS [144, 145]. Thirdly, the challenge for interpretation as rare polymorphism does not necessarily implies functional relevance like for factor H [161] or FB [162] and as mutations in the same gene according to their localization can have different functional consequences on phenotype [163] and even predispose to different pathologies [155]. Complement activation can also be assessed by immunohistochemistry and immunofluorescence, in kidney tissue for examples. Choosing the right antibody according to the raised question is critical as many antibodies recognize several forms of the same protein among native, activated, and degraded. For C3, for example, C3b—its activated form—is inactivated by Factor I into iC3b, unable to form a C3 convertase. Dependently of CR1, FI cleaves iC3b in C3c which is released into circulation, and C3dg which remains bound to the cell and is transformed in C3d by cell proteases [1]. Antibody choice is crucial, as fixation of the tissue is responsible for destruction of C3b neoepitopes which will not recover its conformational state during antigen retrieval. Then, according to the question, a choice will be required between an anti-C3c antibody (which recognizes native C3, C3b, and iC3b, reflecting recent and transient deposition but also local production) and an anti-C3d (C3d is specific and remains long-term attached to cell membrane but will not distinguish an active process). In routine, anti-C3c is mainly used in European countries, risking to underdiagnose semirecent complement-associated pathologies. All these challenges in complement evaluation underline the need for new tools in complement pathologies to make a quick diagnosis, characterize evolution, evaluate response to treatment, and guarantee recovery. This book of methods will provide up-todate protocols according to the most recent knowledge about complement with concise, reliable, and reproducible techniques for complement investigation to answer to this end. However, challenges in exploring complement remain a wide field with many pathways to be investigate, notably regarding the growing number of complement inhibitors. References 1. Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT (2015) Complement system part I—molecular mechanisms of activation and regulation. Front Immunol 6:262 2. Naughton MA et al (1996) Extrahepatic secreted complement C3 contributes to

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inflammasome activity in CD4+ T cells. Science 352:aad1210 19. Ling GS et al (2018) C1q restrains autoimmunity and viral infection by regulating CD8 + T cell metabolism. Science 360:558–563 20. Kremlitzka M et al (2019) Interaction of serum-derived and internalized C3 with DNA in human B cells—a potential involvement in regulation of gene transcription. Front Immunol 10:493 21. Merle NS, Noe R, Halbwachs-Mecarelli L, Fremeaux-Bacchi V, Roumenina LT (2015) Complement system part II: role in immunity. Front Immunol 6:257 22. Pettigrew HD, Teuber SS, Gershwin ME (2009) Clinical significance of complement deficiencies. Ann N Y Acad Sci 1173:108–123 23. Degn SE, Jensenius JC, Thiel S (2011) Disease-causing mutations in genes of the complement system. Am J Hum Genet 88:689–705 24. Rosain J et al (2014) Complement deficiencies and human diseases. Ann Biol Clin (Paris) 72:271–280 25. Brocklebank V, Wood KM, Kavanagh D (2018) Thrombotic Microangiopathy and the kidney. Clin J Am Soc Nephrol 13:300–317 26. Smith RJH et al (2019) C3 glomerulopathy— understanding a rare complement-driven renal disease. Nat Rev Nephrol 15:129–143 27. Corvillo F et al (2019) Nephritic factors: an overview of classification, diagnostic tools and clinical associations. Front Immunol 10:886 28. Marinozzi MC et al (2017) Anti-factor B and anti-C3b autoantibodies in C3 Glomerulopathy and Ig-associated Membranoproliferative GN. J Am Soc Nephrol 28:1603–1613 29. Chauvet S et al (2020) Anti-factor B antibodies and acute Postinfectious GN in children. J Am Soc Nephrol 31(4):829–840. https:// doi.org/10.1681/ASN.2019080851 30. Vasilev VV et al (2019) Autoantibodies against C3b—functional consequences and disease relevance. Front Immunol 10:64 31. Chauvet S et al (2017) Treatment of B-cell disorder improves renal outcome of patients with monoclonal gammopathy–associated C3 glomerulopathy. Blood 129:1437–1447 32. Bu F et al (2016) High-throughput genetic testing for thrombotic Microangiopathies and C3 Glomerulopathies. J Am Soc Nephrol 27:1245–1253 33. Osborne AJ et al (2018) Statistical validation of rare complement variants provides insights into the molecular basis of atypical hemolytic uremic syndrome and C3 Glomerulopathy. J Immunol 200:2464–2478

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47. Roumenina LT et al (2019) Tumor cells hijack macrophage-produced complement C1q to promote tumor growth. Cancer Immunol Res 7:1091–1105 48. Bonavita E et al (2015) PTX3 is an extrinsic oncosuppressor regulating complementdependent inflammation in cancer. Cell 160:700–714 49. Nabizadeh JA et al (2016) The complement C3a receptor contributes to melanoma tumorigenesis by inhibiting neutrophil and CD4+ T cell responses. J Immunol 1950 (196):4783–4792 50. Janelle V et al (2014) Transient complement inhibition promotes a tumor-specific immune response through the implication of natural killer cells. Cancer Immunol Res 2:200–206 51. Bulla R et al (2016) C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation. Nat Commun 7:10346 52. Hajishengallis G, Reis ES, Mastellos DC, Ricklin D, Lambris JD (2017) Novel mechanisms and functions of complement. Nat Immunol 18:1288–1298 53. Carpanini SM, Torvell M, Morgan BP (2019) Therapeutic inhibition of the complement system in diseases of the central nervous system. Front Immunol 10:362 54. Stevens B et al (2007) The classical complement cascade mediates CNS synapse elimination. Cell 131:1164–1178 55. Shi Q et al (2015) Complement C3-deficient mice fail to display age-related hippocampal decline. J Neurosci 35:13029–13042 56. Heppner FL, Ransohoff RM, Becher B (2015) Immune attack: the role of inflammation in Alzheimer disease. Nat Rev Neurosci 16:358–372 57. Brennan FH, Lee JD, Ruitenberg MJ, Woodruff TM (2016) Therapeutic targeting of complement to modify disease course and improve outcomes in neurological conditions. Semin Immunol 28:292–308 58. Hernandez MX, Namiranian P, Nguyen E, Fonseca MI, Tenner AJ (2017) C5a increases the injury to primary neurons elicited by Fibrillar amyloid Beta. ASN Neuro 9:1759091416687871 59. Lambert J-C et al (2009) Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer’s disease. Nat Genet 41:1094–1099 60. Zhou J, Fonseca MI, Pisalyaput K, Tenner AJ (2008) Complement C3 and C4 expression in C1q sufficient and deficient mouse models of

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73. Haines JL et al (2005) Complement factor H variant increases the risk of age-related macular degeneration. Science 308:419–421 74. Seddon JM et al (2013) Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration. Nat Genet 45:1366–1370 75. Weismann D et al (2011) Complement factor H binds malondialdehyde epitopes and protects from oxidative stress. Nature 478:76–81 76. Shaw PX et al (2012) Complement factor H genotypes impact risk of age-related macular degeneration by interaction with oxidized phospholipids. Proc Natl Acad Sci U S A 109:13757–13762 77. van Lookeren Campagne M, Strauss EC, Yaspan BL (2016) Age-related macular degeneration: complement in action. Immunobiology 221:733–739 78. Hughes AE et al (2006) A common CFH haplotype, with deletion of CFHR1 and CFHR3, is associated with lower risk of age-related macular degeneration. Nat Genet 38:1173–1177 79. Cipriani V et al (2020) Increased circulating levels of factor H-related protein 4 are strongly associated with age-related macular degeneration. Nat Commun 11:778 80. Brodsky RA (2014) Paroxysmal nocturnal hemoglobinuria. Blood 124:2804–2811 81. Griffin M et al (2019) Significant hemolysis is not required for thrombosis in paroxysmal nocturnal hemoglobinuria. Haematologica 104:94–96 82. Risitano AM et al (2009) Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab. Blood 113:4094–4100 83. Hill A et al (2010) Eculizumab prevents intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low-level extravascular hemolysis occurring through C3 opsonization. Haematologica 95:567–573 84. Lin Z et al (2015) Complement C3dgmediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria. Blood 126:891–894 85. Wang RH, Phillips G, Medof ME, Mold C (1993) Activation of the alternative complement pathway by exposure of phosphatidylethanolamine and phosphatidylserine on erythrocytes from sickle cell disease patients. J Clin Invest 92:1326–1335 86. Mold C, Tamerius JD, Phillips G (1995) Complement activation during painful crisis

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in sickle cell anemia. Clin Immunol Immunopathol 76:314–320 87. Lombardi E et al (2019) Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule. Haematologica 104:919–928 88. Roumenina LT et al (2020) Complement activation in sickle cell disease: dependence on cell density, hemolysis and modulation by hydroxyurea therapy. Am J Hematol 95 (5):456–464. https://doi.org/10.1002/ajh. 25742 89. Merle NS et al (2018) Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles. JCI Insight 3 (12):e96910 90. Vercellotti GM et al (2019) Critical role of C5a in sickle cell disease. Am J Hematol 94:327–337 91. Merle NS et al (2019) P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/hemedependent manner. Proc Natl Acad Sci 116:6280–6285 92. Merle NS, Boudhabhay I, Leon J, FremeauxBacchi V, Roumenina LT (2019) Complement activation during intravascular hemolysis: implication for sickle cell disease and hemolytic transfusion reactions. Transfus Clin Biol 26:116–124 93. Bork K, Witzke G (1989) Long-term prophylaxis with C1-inhibitor (C1 INH) concentrate in patients with recurrent angioedema caused by hereditary and acquired C1-inhibitor deficiency. J Allergy Clin Immunol 83:677–682 94. Hillmen P et al (2004) Effect of Eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med 350:552–559 95. Hillmen P et al (2006) The complement inhibitor Eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med 355:1233–1243 96. Brodsky RA et al (2008) Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria. Blood 111:1840–1847 97. Hillmen P et al (2013) Long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol 162:62–73 98. Gruppo RA, Rother RP (2009) Eculizumab for congenital atypical hemolytic–uremic syndrome. N Engl J Med 360:544–546 99. Legendre CM et al (2013) Terminal complement inhibitor Eculizumab in atypical

hemolytic–uremic syndrome. N Engl J Med 368:2169–2181 100. Greenbaum LA et al (2016) Eculizumab is a safe and effective treatment in pediatric patients with atypical hemolytic uremic syndrome. Kidney Int 89:701–711 101. Percheron L et al (2018) Eculizumab treatment in severe pediatric STEC-HUS: a multicenter retrospective study. Pediatr Nephrol 33:1385–1394 102. Burwick RM, Feinberg BB (2013) Eculizumab for the treatment of preeclampsia/ HELLP syndrome. Placenta 34:201–203 103. de Fontbrune FS et al (2015) Use of Eculizumab in patients with allogeneic stem cell transplant-associated thrombotic Microangiopathy: a study from the SFGM-TC. Transplantation 99:1953–1959 104. Fakhouri F et al (2016) Terminal complement inhibitor Eculizumab in adult patients with atypical hemolytic uremic syndrome: a single-arm, open-label trial. Am J Kidney Dis 68:84–93 105. Nishimura J et al (2014) Genetic variants in C5 and poor response to Eculizumab. N Engl J Med 370:632–639 106. Risitano AM et al (2019) Anti-complement treatment for paroxysmal nocturnal hemoglobinuria: time for proximal complement inhibition? A position paper from the SAAWP of the EBMT. Front Immunol 10:1157 107. Rawal N, Pangburn MK (2000) Functional role of the noncatalytic subunit of complement C5 convertase. J Immunol 164:1379–1385 108. Ardissino G et al (2015) Discontinuation of Eculizumab treatment in atypical hemolytic uremic syndrome: an update. Am J Kidney Dis 66:172–173 109. Merrill SA et al (2017) Eculizumab cessation in atypical hemolytic uremic syndrome. Blood 130:368–372 110. Fakhouri F et al (2017) Pathogenic variants in complement genes and risk of atypical hemolytic uremic syndrome relapse after Eculizumab discontinuation. Clin J Am Soc Nephrol 12:50–59 111. Ricklin D, Mastellos DC, Reis ES, Lambris JD (2018) The renaissance of complement therapeutics. Nat Rev Nephrol 14:26–47 112. Tatapudi VS, Montgomery RA (2019) Therapeutic modulation of the complement system in kidney transplantation: clinical indications and emerging drug leads. Front Immunol 10:2306 113. Viglietti D et al (2016) C1-inhibitor in acute antibody-mediated rejection non-responsive

Complement in the Clinical Practice to conventional therapy in kidney transplant recipients: a pilot study. Am J Transplant 16 (5):1596–1603. https://doi.org/10.1111/ ajt.13663 114. Montgomery RA et al (2016) Plasma-derived C1 esterase inhibitor for acute antibodymediated rejection following kidney transplantation: results of a randomized doubleblind placebo-controlled pilot study. Am J Transplant 16:3468–3478 115. Stegall MD et al (2011) Terminal complement inhibition decreases antibody-mediated rejection in sensitized renal transplant recipients. Am J Transplant 11:2405–2413 116. Park DH, Connor KM, Lambris JD (2019) The challenges and promise of complement therapeutics for ocular diseases. Front Immunol 10:1007 117. Pittock SJ et al (2019) Eculizumab in Aquaporin-4–positive Neuromyelitis Optica Spectrum disorder. N Engl J Med 381:614–625 118. Karasu E, Nilsson B, Ko¨hl J, Lambris JD, Huber-Lang M (2019) Targeting complement pathways in Polytrauma- and sepsisinduced multiple-organ dysfunction. Front Immunol 10:543 119. Thurman JM, Yapa R (2019) Complement therapeutics in autoimmune disease. Front Immunol 10:672 120. Pickering MC et al (2015) Eculizumab as rescue therapy in severe resistant lupus nephritis. Rheumatology (Oxford) 54:2286–2288 121. Park MH, Caselman N, Ulmer S, Weitz IC (2018) Complement-mediated thrombotic microangiopathy associated with lupus nephritis. Blood Adv 2:2090–2094 122. Howard JF et al (2017) Safety and efficacy of eculizumab in anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis (REGAIN): a phase 3, randomised, double-blind, placebo-controlled, multicentre study. Lancet Neurol 16:976–986 123. Lonze BE, Singer AL, Montgomery RA (2010) Eculizumab and renal transplantation in a patient with CAPS. N Engl J Med 362:1744–1745 124. Shapira I, Andrade D, Allen SL, Salmon JE (2012) Brief report: induction of sustained remission in recurrent catastrophic antiphospholipid syndrome via inhibition of terminal complement with eculizumab. Arthritis Rheum 64:2719–2723 125. Jayne DRW et al (2017) Randomized trial of C5a receptor inhibitor Avacopan in ANCA-

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associated Vasculitis. J Am Soc Nephrol 28:2756–2767 126. Pio R, Ajona D, Ortiz-Espinosa S, Mantovani A, Lambris JD (2019) Complementing the cancer-immunity cycle. Front Immunol 10:774 127. Crew PE et al (2020) Antibiotic prophylaxis in vaccinated eculizumab recipients who developed meningococcal disease. J Infect 80:350–371 128. Mohebnasab M et al (2019) Current and future approaches for monitoring responses to anti-complement therapeutics. Front Immunol 10:2539 129. He Y et al (2020) Normal range of complement components during pregnancy: a prospective study. Am J Reprod Immunol 83: e13202 130. Ekdahl KN et al (2018) Interpretation of serological complement biomarkers in disease. Front Immunol 9:2237 131. Nilsson PH et al (2017) Eculizumab-C5 complexes express a C5a neoepitope in vivo: consequences for interpretation of patient complement analyses. Mol Immunol 89:111–114 132. Platts-Mills TA, Ishizaka K (1974) Activation of the alternate pathway of human complements by rabbit cells. J Immunol 1950 (113):348–358 133. Yamamoto S et al (1995) Automated homogeneous liposome-based assay system for total complement activity. Clin Chem 41:586–590 134. Seelen MA et al (2005) Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA. J Immunol Methods 296:187–198 135. Gavriilaki E et al (2015) Modified ham test for atypical hemolytic uremic syndrome. Blood 125:3637–3646 136. Noris M et al (2014) Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood 124:1715–1726 137. Palomo M et al (2019) Complement activation and thrombotic Microangiopathies. Clin J Am Soc Nephrol 14:1719–1732 138. Daha MR, Fearon DT, Austen KF (1976) C3 nephritic factor (C3NeF): stabilization of fluid phase and cell-bound alternative pathway convertase. J Immunol 1950(116):1–7 139. Servais A et al (2012) Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 82:454–464

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140. Marinozzi M-C et al (2017) C5 nephritic factors drive the biological phenotype of C3 glomerulopathies. Kidney Int 92:1232–1241 141. Halbwachs L, Leveille´ M, Lesavre P, Wattel S, Leibowitch J (1980) Nephritic factor of the classical pathway of complement: immunoglobulin G autoantibody directed against the classical pathway C3 convertase enzyme. J Clin Invest 65:1249–1256 142. Gigli I, Sorvillo J, Mecarelli-Halbwachs L, Leibowitch J (1981) Mechanism of action of the C4 nephritic factor. Deregulation of the classical pathway of C3 convertase. J Exp Med 154:1–12 143. Zhang Y et al (2017) C4 nephritic factors in C3 Glomerulopathy: a case series. Am J Kidney Dis 70:834–843 144. Zipfel PF et al (2007) Deletion of complement factor H–related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome. PLoS Genet 3:e41 145. Dragon-Durey M-A et al (2009) The high frequency of complement factor H related CFHR1 gene deletion is restricted to specific subgroups of patients with atypical haemolytic uraemic syndrome. J Med Genet 46:447–450 146. Chen Q et al (2011) Combined C3b and factor B autoantibodies and MPGN type II. N Engl J Med 365:2340–2342 147. Grumach AS, Kirschfink M (2014) Are complement deficiencies really rare? Overview on prevalence, clinical importance and modern diagnostic approach. Mol Immunol 61:110–117 148. El Sissy C et al (2019) Clinical and genetic Spectrum of a large cohort with Total and sub-total complement deficiencies. Front Immunol 10:1936 149. Fremeaux-Bacchi V et al (2013) Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults. Clin J Am Soc Nephrol 8:554–562 150. Noris M et al (2010) Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol 5:1844–1859 151. Toomey CB, Johnson LV, Bowes Rickman C (2018) Complement factor H in AMD: bridging genetic associations and pathobiology. Prog Retin Eye Res 62:38–57

152. Servais A et al (2007) Primary glomerulonephritis with isolated C3 deposits: a new entity which shares common genetic risk factors with haemolytic uraemic syndrome. J Med Genet 44:193–199 153. Zipfel PF et al (2015) The role of complement in C3 glomerulopathy. Mol Immunol 67:21–30 154. Quintrec ML et al (2013) Complement genes strongly predict recurrence and graft outcome in adult renal transplant recipients with atypical hemolytic and uremic syndrome. Am J Transplant 13:663–675 155. de Cordoba SR, Tortajada A, Harris CL, Morgan BP (2012) Complement dysregulation and disease: from genes and proteins to diagnostics and drugs. Immunobiology 217:1034–1046 156. Heinen S et al (2006) De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome. Hum Mutat 27:292–293 157. Francis NJ et al (2012) A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome. Blood 119:591–601 158. de Jorge EG et al (2018) Factor H Competitor Generated by Gene Conversion Events Associates with Atypical Hemolytic Uremic Syndrome. J Am Soc Nephrol 29:240–249 159. Xiao X et al (2016) Familial C3 glomerulonephritis caused by a novel CFHR5-CFHR2 fusion gene. Mol Immunol 77:89–96 160. Gale DP et al (2010) Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis. Lancet 376:794–801 161. Tortajada A et al (2012) Complement factor H variants I890 and L1007 while commonly associated with atypical hemolytic uremic syndrome are polymorphisms with no functional significance. Kidney Int 81:56–63 162. Marinozzi MC et al (2014) Complement factor B mutations in atypical hemolytic uremic syndrome-disease-relevant or benign? J Am Soc Nephrol 25:2053–2065 163. Roumenina LT et al (2012) A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function. Blood 119:4182–4191

Chapter 2 Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source Seline A. Zwarthoff, Simone Magnoni, Piet C. Aerts, Kok P. M. van Kessel, and Suzan H. M. Rooijakkers Abstract Understanding how human complement proteins interact with human antibodies is important for the development of antibody therapies and understanding autoimmune diseases. At present, many groups use baby rabbit serum as a source of complement because, in contrast to human serum, it lacks preexisting antibodies. However, for characterization of human (monoclonal) antibodies, human serum would be a preferred source of complement. To prevent complement activation via naturally occurring antibodies, this human serum ideally lacks IgG and IgM. Here we describe how to deplete human serum of naturally occurring IgG and IgM using fast protein liquid affinity chromatography (FPLC) while minimizing the loss of serum complement activity. We also describe assays that can be used to validate depletion of IgG and IgM (IgG, IgM, and C1q sandwich ELISAs) and functionally assess remaining serum complement activity (hemolytic assays CH50 and AH50). Finally, we demonstrate how captured IgG and IgM can be purified. Key words Complement, Classical pathway, IgG depletion, IgM depletion, Affinity chromatography, Immunoassay, AH50, AP50, CH50, Antibody therapy

1

Introduction The co1mplement system is a conserved group of serum immune proteins important for host protection against invading microbes. Human IgG and IgM antibodies play an important role in the activation of the complement cascade. When these antibodies recognize antigens on a cell surface, the first complement protein C1 can bind their Fc domains. This initiates a proteolytic cascade that results in opsonization of the target cell surface, release of anaphylatoxins, and lysis of the cell via a lytic multiprotein pore, the membrane attack complex (MAC). Although complement is a powerful defense mechanism, imbalanced or unwanted complement activation can cause autoimmune reactions. In order to develop potent antibody therapies against infectious agents and/or understand autoimmune diseases, it is important to better

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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understand the mechanisms underlying the antibody-mediated complement activation. Baby rabbit serum lacks preexisting antibodies and is frequently used as a source of complement in studies with human antibodies. However, human serum would be the most natural and preferred source of complement [1, 2]. Human serum typically contains 7–18 mg IgG and 0.4–4 mg IgM per mL serum [3]. Evaluation of complement activation by a well-defined antibody such as monoclonal antibodies can be difficult due to presence of these naturally occurring antibodies. For the functional analyses of human antibodies, human serum lacking natural IgG and IgM antibodies is thus very useful. Here we describe how to deplete human serum of IgG and IgM using fast protein liquid affinity chromatography (FPLC). Although protocols for depletion of IgG via Protein G affinity chromatography have been described before, to our knowledge these have not been combined with IgM depletion [4]. IgG and IgM molecules were captured by affinity chromatography using Protein G and CaptureSelect IgM affinity matrix respectively. We describe specific precautions to minimize the loss of serum complement activity (since complement is easily activated and consumed during the FPLC procedure), and demonstrate how to quantify antibody and (functional) complement levels after antibody depletion in ELISAs and classical pathway and alternative pathway hemolytic assays (CH50 and AH50). Finally, we show how to recover the captured natural IgG and IgM from the columns.

2

Materials Unless otherwise stated, we use ultrapure water (type 1) when referring to water. Buffers described below are filter-sterilized (
0.45 μm) after preparation.

2.1 IgG and IgM Depletion by FLPC

1. AKTA FPLC system or equivalent. 2. IgM-binding column: 10 mL POROS™ CaptureSelect™ IgM Affinity Matrix (Thermo Scientific™) with theoretical binding capacity of 5 mg IgM per mL resin, poured into an empty column (XK column, GE Healthcare). 3. IgG-binding column: Prepacked 5 mL HiTrap Protein G High Performance column (GE Healthcare) with theoretical binding capacity of 25 mg IgG per mL. 4. Dialysis tubing (MWCO 14,000 Da). 5. Human serum (see Note 1). 6. Ethylenediaminetetraacetic acid (EDTA, 0.5 M): Add 18.61 g of EDTA-2Na·2H2O to 50 mL of water. Adjust pH to 8.0 with

Depletion of IgG and IgM from Human Serum

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NaOH (this is required to dissolve the EDTA) and make up to 100 mL with water. 7. Phosphate-buffered saline (PBS): 2.7 mM KCl, 1.5 mM KH2PO4, 138 mM NaCl, 6.9 mM Na2HPO4·2H2O. Dissolve 2 g KCl, 2 g KH2PO4, 80 g NaCl, 12.3 g Na2HPO4·2H2O in 10 L water. Adjust pH to 7.1–7.3. 8. Calcium chloride (1 M): Dissolve 7.35 g CaCl2·2H2O in 100 mL water. Store at 4  C. 9. Magnesium chloride (1 M): Dissolve 10.37 g MgCl2·6H2O in 100 mL water. Store at 4  C. 10. Elution buffer: 100 mM glycine–HCl, pH 2.7. Dissolve 7.507 g glycine in 800 mL of water. Adjust pH to 2.5 with HCl. Make up to 1 L with water. Store at 4  C but bring to room temperature for use. 11. Tris (1 M): Dissolve 12.11 g Tris in 80 mL water. Adjust pH to 10.5. Make up to 100 mL with water. 12. Storage buffer: PBS containing 0.1% NaN3 (w/v): Dissolve 100 mg of NaN3 in 100 mL PBS. 2.2

ELISA

1. Absorbance microplate reader. 2. Nunc MaxiSorp 96-well flat bottom microtiter plates. 3. Coating antibodies: sheep anti-human IgG (682311, ICN Biomedicals) (IgG ELISA), sheep anti-human IgM (682331, ICN Biomedicals) (IgM ELISA), mouse anti-C1q antibody (A201, Quidel) (C1q ELISA). 4. Detection antibodies: HRP-conjugated goat anti-human IgG (2040-05, Southern Biotech) (IgG ELISA), HRP-conjugated goat anti-human IgM (2020-05, Southern Biotech) (IgM ELISA), rabbit anti-human C1q (A0136, Dako) (C1q ELISA), HRP-conjugated goat anti-rabbit IgG (4030-05, Southern Biotech) (C1q ELISA) (see Note 2). 5. Sodium carbonate buffer (0.1 M): Prepare a 0.1 M NaHCO3 solution by dissolving 840 mg NaHCO3 in 100 mL water. Prepare a 0.1 M Na2CO3·10H2O solution by dissolving 2.86 g Na2CO3·10H2O in 100 mL water. Mix both solutions until pH is 9.6 to create the sodium carbonate buffer. 6. PBS-T: PBS containing 0.05% Tween 20 (v/v). Dilute Tween 202,000 in PBS. 7. Blocking buffer A: PBS-T containing 4% bovine serum albumin (BSA) (bovine albumin fraction V pH 7.0, Serva) (w/v). Dissolve 400 mg BSA per 10 mL PBS-T. Prepare fresh. 8. Blocking buffer B: PBS-T containing 3% skimmed milk powder (w/v). Dissolve 300 mg skimmed milk powder per 10 mL PBS-T. Prepare fresh.

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9. Dilution buffer A: PBS-T containing 1% bovine serum albumin (BSA) (w/v). Dilute Blocking buffer A 4 in PBS-T. 10. Dilution buffer B: PBS-T containing 1% skimmed milk powder (w/v). Dilute Blocking buffer B 3 in PBS-T. 11. Sodium acetate (1.1 M): dissolve 9 g of sodium acetate in 80 mL water. Adjust pH to 6 with 15 N citric acid and make up to 100 mL with water. Store at 4  C. 12. 3,30 ,5,50 -Tetramethylbenzidine (TMB, 6 mg/mL): Dissolve 6 mg of TMB per 1 mL DMSO. Store in the dark at room temperature. 13. Urea hydrogen peroxide (20 mg/mL): Dissolve 200 mg of urea hydrogen peroxide in 10 mL water. Store at 4  C. 14. ELISA substrate: 0.11 M sodium acetate, 0.1 mg/mL TMB, 0.17 mg/mL urea hydrogen peroxide. Add 600 μL 1.1 M sodium acetate, 100 μL TMB, and 50 μL urea hydrogen peroxide to 5.4 mL of water. Prepare the substrate just before use. 15. H2SO4 (1 N): Add 2.8 mL of 36 N H2SO4 to 97.2 mL of water. Take safety precautions when working with highly concentrated H2SO4 solutions. 2.3 Hemolytic Assays

1. Optical density meter for tubes. 2. Absorbance microplate reader. 3. 96-well round bottom microtiter plates. 4. 96-well flat bottom microtiter plates. 5. Sheep blood in Alsever solution (bioTrading). 6. Rabbit anti-sheep RBC amboceptor. In-house rabbit antiserum obtained by plasmapheresis after 6 immunization (7-day intervals, first immunization including Freund’s Complete Adjuvant) with 20% washed sheep red blood cells (bioTrading). 7. Rabbit blood in Alsever solution (bioTrading). 8. Phosphate-buffered saline (PBS): see above. 9. Veronal buffer (VBS; 5): 727 mM NaCl, 9.1 mM sodium barbital, 10.2 mM 5,5-diethyl barbituric acid, pH 7.4–7.6. To prepare 1 L, dissolve 42.5 g NaCl and 1.875 g sodium barbital in 700 mL water. Dissolve 1.875 g 5,5-diethylbarbituric acid in 100 mL warm water and add to the NaCl/barbital solution. Adjust pH to 7.4–7.6 and make up to 1 L with water. 10. VBS++: Veronal buffer containing 0.5 mM CaCl2, 0.25 mM MgCl2. Mix 10 mL 5 Veronal buffer with 25 μL 1 M CaCl2 and 12.5 μL 1 M MgCl2. Make up to 50 mL with water. 11. MgEGTA (10): 50 mM MgCl2, 100 mM EGTA, pH 7.5. Add 3.8 g ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA) and 5 mL 1 M MgCl2 to 50 mL

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water. Adjust pH to 8 with NaOH (this is required to dissolve the EGTA) and make up to 100 mL with water. 12. VBS-MgEGTA: Veronal buffer containing 0.5 mM MgCl2, 10 mM EGTA. Mix 10 mL 5 Veronal buffer with 5 mL 10 MgEGTA, make up to 50 mL with water.

3

Methods

3.1 IgG and IgM Depletion by Affinity Chromatography

1. Draw blood from healthy humans and collect in glass vacutainer (see Note 1). Allow to clot for 15 min at room temperature and centrifuge blood at 3166  g for 10 min at 4  C to collect serum (supernatant). Pool collected sera and keep serum on ice during all following procedures to minimize complement activation unless otherwise stated or not possible (see Note 3). 2. For affinity chromatography with the proposed IgG- and IgM-binding columns, take 5 mL serum and add 25 μL EDTA to prevent complement activation by the column materials (see Notes 4 and 5). 3. Couple the CaptureSelect column and HiTrap column in tandem (CaptureSelect column first) and connect to AKTA-FPLC system. Wash columns with 50 mL ice cold PBS. 4. Load 5 mL serum in the AKTA-FPLC sample loop. Inject 5 mL serum at a flow speed of 1 mL/min over both columns and collect 0.5 mL fractions. Pool serum fractions by visual inspection for the most serum-like appearance (yellow) and peak in UV absorbance profile (see Note 6). 5. Determine pooled volume and add 5 μL 1 M CaCl2 and 5 μL 1 M MgCl2 per mL serum to reconstitute the ions. Directly aliquot serum and store at 80  C (see Note 7).

3.2 Recovery of Bound IgG and IgM from Column

1. IgG and IgM antibodies can be eluted as functional proteins from each column separately (see Note 8). Connect one column to AKTA-FPLC and wash the column with 5 column volume (CV) PBS. 2. Elute bound IgG or IgM with 5 CV Elution buffer and collect 0.5 mL fractions. Neutralize collected fractions immediately with 30 μL Tris. Pool the fractions containing eluted protein based on the peak UV 280 nm absorbance and dialyze the pooled fractions of IgG or IgM overnight against cold PBS. 3. Measure the 280 nm absorbance of the eluted IgG or IgM and calculate the protein concentration using an absorbance coefficient of 1.36 for IgG or 1.18 for IgM. 4. Wash both columns with 5 CV PBS. Store the columns in Storage buffer at 4  C.

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IgG ELISA

1. To coat the plate, fill wells in a 96-well Nunc MaxiSorp microtiter plate with 50 μL of sheep anti-human IgG (1:1000 dilution or 2 μg/mL in sodium carbonate buffer) and incubate overnight at 4  C. 2. Wash the wells 3 with 200 μL PBS-T (see Note 9). 3. Block wells with 80 μL of Blocking buffer A for 1 h at 37  C. 4. Wash the wells 3 with 200 μL PBS-T. 5. Prepare serial dilutions of depleted and normal serum in Dilution buffer A and include a buffer control (see Note 10). Fill the wells with 50 μL serial dilution of the depleted serum or normal serum. Incubate for 1 h at 37  C. 6. Wash the wells 3 with 200 μL PBS-T. 7. For detection, incubate wells with 50 μL goat anti-human IgG-HRP (1:6000 diluted in Dilution buffer A) for 1 h at 37  C. 8. Wash the wells 3 with 200 μL PBS-T. 9. Incubate wells with 50 μL ELISA Substrate for 5–10 min at room temperature. 10. Stop the reaction by adding 50 μL H2SO4 to each well. Directly measure 450 nm absorbance in a microplate reader. The percentage of residual IgG in the depleted serum can be calculated by comparing the half-maximal effective concentration (EC50) of depleted and normal serum. When expressing the EC50 as serum dilution factor, the residual IgG level (%) ¼ [EC50depleted serum/EC50normal serum]  100. The percentage of depletion of IgG ¼ 100  Residual IgG (%) (see Note 11).

3.4

IgM ELISA

As for IgG ELISA, but with the following changes. Use sheep antihuman IgM (1:1000 dilution or 2 μg/mL in sodium carbonate buffer) to coat the plate. Use goat anti-human IgM-HRP (1:6000 dilution in Dilution buffer A) for detection (see Note 11).

3.5

C1q ELISA

As for IgG ELISA, but with the following changes. Use mouse antiC1q antibody (1:1000 dilution or 1 μg/mL in sodium carbonate buffer) to coat the plate. Use Blocking buffer B to block the plate. After blocking, use Dilution buffer B for all following serum and antibody dilutions. For detection, incubate wells with 50 μL rabbit anti-C1q antibody (1:1000 in Dilution buffer B) for 1 h at 37  C. Wash the wells 3 with 200 μL PBS-T. Next, incubate wells with 50 μL goat anti-rabbit-IgG-HRP (1:6000 in Dilution buffer B) for 1 h at 37  C. Wash wells 3 with 200 μL PBS-T. Then add ELISA substrate and continue protocol as described for IgG ELISA (see Note 11).

Depletion of IgG and IgM from Human Serum

3.6 Classical Pathway Hemolytic Assay (CH50)

27

1. Pipet 1 mL sheep blood in a 5 mL tube and wash this 3 with 1 mL PBS at 1780  g for 5 min. Vortex the pellet between washes (see Note 12). 2. Vortex the pellet of erythrocytes and prepare a 1:25 dilution by adding 100 μL erythrocytes to 2400 μL VBS++. Gently invert the tube to mix. To check the concentration of the erythrocytes, transfer 40 μL erythrocytes to 1960 μL water (1:50 dilution) to lyse the cells and measure the OD at 405 nm. The OD405 should be around 0.52, if not, change the dilution factor of the erythrocytes in VBS++ and do the check again. 3. Sensitize erythrocytes by mixing 2 mL of the erythrocytes in VBS++ with 2 mL rabbit IgM anti-sheep RBC (diluted 1:1000 in VBS++). Leave at room temperature for 10 min (no shaking). 4. Wash the sensitized erythrocytes 1 with 2 mL VBS++ at 1780  g for 5 min. Resuspend the pellet in 4 mL VBS++. 5. In a round bottom plate, add 100 μL two-fold serial dilutions of depleted and normal serum in VBS++, starting at a dilution of 1:10 (see Note 13). Also fill one well with 100 μL water (positive control) and one well with 100 μL VBS++ (negative control). 6. Add 50 μL sensitized erythrocytes to all samples. Incubate for 45 min at 37  C. 7. Spin down plate at 1780  g for 10 min. Transfer 50 μL of the supernatant to 100 μL water in a new flat bottom 96-well plate, mix well and measure hemolysis at OD405 nm in the plate reader. Calculate the percentage of lysis for each sample as follows: [OD405sample  OD405neg.ctrl.]/[OD405pos. ctrl.  OD405neg.ctrl.]  100. Plot the percentage of lysis against the serum concentration (dilution factor) and calculate the dilution factor of serum required for 50% lysis (¼CH50). The residual CH50 activity (%) of depleted serum ¼ [CH50depleted serum/CH50normal serum]  100 (see Note 11).

3.7 Alternative Pathway Hemolytic Assay (AH50)

1. Pipet 1 mL rabbit blood in a 5 mL tube and wash this 3 with 1 mL PBS at 1780  g for 5 min. Vortex the pellet between washes (see Note 12). 2. Vortex the pellet of erythrocytes and prepare a 1:25 dilution by adding 100 μL erythrocytes to 2400 μL VBS-MgEGTA. Gently invert the tube to mix (see Note 14). To check the concentration of the erythrocytes, transfer 100 μL erythrocytes to 2400 μL water (1:25 dilution) to lyse the cells and measure the OD at 405 nm. The OD405 should be around 1.23, if not, change the dilution factor of the erythrocytes in VBS-MgEGTA and do the check again.

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3. In a round bottom plate, add 100 μL 1.5-fold serial dilutions of depleted and normal serum in VBS-MgEGTA, starting at a concentration of 1:6.7 (see Note 13). Also fill one well with 100 μL water (positive control) and one well with 100 μL VBS-MgEGTA (negative control). 4. Add 50 μL erythrocytes to all samples. Incubate for 45 min at 37  C. 5. Spin down plate at 1780  g for 10 min. Transfer 50 μL of the supernatant to 100 μL water in a new flat bottom 96-well plate, mix well and measure hemolysis at OD405 nm in the plate reader. Calculate the percentage of lysis for each sample as follows: [OD405sample  OD405neg.ctrl.]/[OD405pos. ctrl.  OD405neg.ctrl.]  100. Plot the percentage of lysis against the serum concentration (dilution factor) and calculate the dilution factor of serum required for 50% lysis (¼AH50). The residual AH50 activity (%) of depleted serum ¼ [AH50depleted serum/AH50normal serum]  100 (see Note 11).

4

Notes 1. Human serum is commercially available but it is often not clear how the serum has exactly been handled and stored. We therefore find it best to isolate serum from human blood ourselves as described below. This allows us to minimize complement activation during the procedure. 2. We chose for a two-step detection of C1q but there are also HRP-conjugated anti-C1q antibodies available. 3. Serum can be stored at 80  C. To prevent complement activation, minimize freeze–thaw cycles and store in aliquots. 4. The theoretical binding capacity of the HiTrap column and CaptureSelect column is 125 mg IgG and 50 mg IgM, respectively. However, in practice especially the binding capacity of the Capture Select column is much lower. For optimal IgM depletion, we found that no more than 5 mL serum should be run over the columns. To deplete larger volumes of serum in the same run, one could use larger or extra columns. Serum that is depleted using the depletion procedure presented here usually contains no detectable IgG and less than 0.5% residual IgM. 5. Calcium and magnesium ions are required for activation of the complement system. To prevent complement activation against the column materials, we treat serum with an excess EDTA

Depletion of IgG and IgM from Human Serum

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which chelates calcium and magnesium ions. After chromatography we reconstitute the serum with sufficient CaCl2 and MgCl2 to restore proper ion concentrations for complement activity. 6. To minimize dilution of the depleted serum, we do not include fractions with a very light yellow appearance in the pool as these contain more PBS. Therefore, we typically recover 70–80% of the input volume (here 3.5–4.0 mL of 5 mL input). 7. IgG depletion typically results in partial codepletion of C1q (Fig. 1), as described in previous studies [4]. The residual C1q

Fig. 1 Quantification of IgG, IgM, and C1q levels by ELISA in normal serum and serum depleted of IgG and IgM using the described procedure. Data shows threefold serial dilutions of normal and depleted (ΔIgGΔIgM) serum starting from 1:9000 and 1:30 dilutions, respectively. The EC50 values and residual protein levels calculated from the ELISA results are shown in Table 1. The data shown are a representative of more than three independent depletion runs. (a) Quantification of IgG levels in an IgG ELISA. No IgG is detected in depleted serum diluted 30-fold or more. (b) Quantification of IgM levels in an IgM ELISA. A low level of IgM is still detected in 30-fold diluted depleted serum. (c) Quantification of C1q levels in a C1q ELISA. C1q is codepleted, but the ΔIgGΔIgM serum still contains about 8% residual C1q

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levels in ΔIgGΔIgM serum vary between depletion runs but are typically around 10% (Fig. 1c). The C1q levels in ΔIgGΔIgM serum could however be restored with (commercially available) C1q to physiological levels before freezing it. In the functional complement activity assays CH50 and AH50 (also known as AP50), the ΔIgGΔIgM serum typically shows 50% residual hemolytic activity, which indicates that other complement factors are not severely depleted by the described procedure. 8. To be able to reuse the columns, captured proteins must be removed. The buffers typically proposed by manufacturers for this cleaning procedure are often of low pH and will severely affect the functionality of the eluted proteins. We here describe how functional antibodies can be eluted from the columns and be used for, for example, reconstitution of the depleted serum. 9. To remove fluid easily from the wells between washes, smash the plate gently but firmly upside-down on a pile of paper tissues. Repeat once or twice until most fluid is gone. 10. We typically find useful S-shaped ELISA curves when testing threefold serial dilutions of serum (8 steps), starting from a 1:30 dilution of depleted serum and 1:9000 dilution of normal serum. If this is not the case, the dilution series should be adjusted. 11. Representative ELISA, CH50, and AH50 results from a typical depletion run are shown in Figs. 1, 2, and Table 1. 12. The pellet can be stored at 4  C for 1 day. 13. Unlike sheep erythrocytes, rabbit erythrocytes are activating surfaces for the alternative pathway and need no sensitization with antibodies. EGTA is added to prevent classical pathway activation by chelating calcium. 14. Usually the concentration of serum used in hemolytic assays is expressed in percentages. However, one can dispute whether the obtained depleted serum should be referred to as 100% serum, since it is slightly diluted in PBS during the depletion procedure and shows reduced complement activity compared to normal serum. We therefore rather describe the dilution factor than percentage of the depleted serum.

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Fig. 2 Quantification of complement activity by classical pathway and alternative pathway specific hemolytic assays (CH50 and AH50, respectively) in normal serum and serum depleted of IgG and IgM using the described procedure. Data shows two-fold serial dilutions of normal and depleted (ΔIgGΔIgM) serum starting from 1:10 (CH50) or 1:6.7 (AH50) dilutions. The EC50 values and residual hemolytic activity levels calculated from the CH50 and AH50 results are shown in Table 1. The data shown are a representative of more than three independent depletion runs. (a) Classical pathway hemolytic assay (CH50) showing the functional capability of the classical pathway complement components in serum to lyse antibody-coated sheep red blood cells. The red blood cell lysis is quantified by the absorbance of released hemoglobin (OD405). (b) Alternative pathway hemolytic assay (AH50 or AP50) showing the functional capability of the alternative pathway complement components in serum to lyse rabbit red blood cells. The red blood cell lysis is quantified by the absorbance of released hemoglobin (OD405) Table 1 EC50 values and residual levels IgG, IgM, and C1q, and hemolytic activity calculated from the ELISA, CH50, and AH50 data shown in Figs. 1 and 2 EC50 (dilution factor) ΔIgGΔIgM

Assay

Normal

Residual level (%)

IgG ELISA

217,741

500 mL buffer by mixing the alkaline 0.15 M acetate solution with the acidic 0.15 M acetic acid solution to pH 4.0. As a rule use approximately 450 mL of the acidic solution for every 100 mL of the alkaline solution.

Quantification of Porcine C3a

2.2 Working Solutions

53

1. PBS with 0.1% Tween 20. Transfer 900 mL PBS into a 1 L volumetric flask. Add 1 mL Tween 20 (see Note 3) and fill up with PBS. Store the buffer in room temperature. 2. PBS with 0.2% Tween 20. Transfer 900 mL PBS into a 1 L volumetric flask. Add 2 mL Tween 20 (see Note 3) and fill up with PBS. Store the buffer in room temperature. 3. PBS with 0.2% Tween 20 and 10 mM EDTA. Transfer 900 mL PBS into a 1 L volumetric flask. Add 2 mL Tween 20 (see Note 3), 20 mL 0.5 M EDTA pH 7.4 and fill up with PBS. This buffer shall be kept cold until use. 4. ABTS (2,20 -azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt) substrate solution. Add 90 mg ABTS to an Erlenmeyer flask. Fill up with 450 mL 0.15 M sodium acetate buffer pH 4.0. Transfer to a 500 mL volumetric flask and fill up with 0.15 M sodium acetate buffer pH 4.0. Protect from light exposure. This buffer should be equilibrated to room temperature before use. 5. 1 M H2SO4 (optional). 6. H2O2 (3%). Dilute 30% H2O2 1/10 in water. This solution must be stored dark and can then be kept for up to 1 week.

2.3

Antibodies

1. Mouse-IgG2bk anti-porcine C3a/C3a desArg (clone Z22/8, Cat. No.: EGO008; Kerafast, Boston, MA). 2. Mouse-IgG1/k anti-porcine C3a/C3a desArg (clone K5/9, Cat. No.: EGO009; Kerafast, Boston, MA). 3. Goat anti-mouse IgG1-HRP (Cat. No.: 1070-05; Southern Biotech. Birmingham, AL).

2.4 Samples, Standards, and Controls

Plasma samples to detect complement activation products generated in vivo, should be obtained from EDTA-blood, and prepared and stored under strict conditions in order to avoid in vitro activation [4, 5] (see Note 4). All handling of EDTA-blood tubes before centrifugation and plasma after centrifugation should be carried out on crushed ice (see Note 5). Samples should be stored at 70
C or colder (see Note 6). The standard curve was created from recombinant porcine C3a (Cat. No.: RP1132S, Kingfisher Biotech, Inc., Saint Paul, MN) (Fig. 1). An optimal curve was obtained by two-fold dilution from 50 ng/mL to 0.39 ng/mL. Normal porcine plasma was found to have an optimal dilution factor of 1/20, giving a mean value for C3a of 37 mg/mL (as indicated by the arrow on Fig. 1). A positive and negative control should be included in every assay (see Note 7). The negative control should be a plasma sample with a low level of activation and the positive control should be a sample with a high level of activation, for example serum incubated with a complement activator (Tables 1 and 2). Serum can be

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Fig. 1 A representative standard curve of recombinant porcine C3a. C3a was diluted to 50 ng/mL and further in twofold dilutions. Absorbance values at 405 nm are plotted against the C3a-concentration on a logarithmic x-axis. A sigmoidal four-parametric standard curve was fitted to the plotted standard values. The standard points were analyzed in duplicate, standard deviation is represented by the error bars. The arrow indicates the point on the standard curve corresponding to the value of the normal porcine plasma diluted 1/20 Table 1 Intra-assay variation Sample NPPe E.c APS

f

Sample activationa

nb

Dilution factor

Mean C3a (ng/mL)c

CVd (%)

No

20

20

37

7.1

Yes

9

200

2224

3.7

a

In vitro sample activation Number of observations c C3a after dilution factor correction d Coefficient of variation e Normal porcine plasma f E. coli–activated porcine serum b

Table 2 Interassay variation Sample

Sample activationa

nb

Dilution factor

Mean C3a (ng/mL)c

CVd (%)

NPPe

No

9

20

37

14

Yes Yes

7 7

800 12,800

1452 27,020

10 9.2

f

ZAPS CAPSg a

In vitro sample activation Number of observations c C3a after dilution factor correction d Coefficient of variation e Normal porcine plasma f Zymosan-activated porcine serum g Cobra venom factor–activated porcine serum b

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55

activated by addition of, for example, cobra venom factor or zymosan. Here, we incubated 20 units of cobra venom factor per mL serum for 90 min at 37
C to fully activate C3. A modest and medium level of C3-activation was achieved by activating complement serum with either zymosan (10 mg/mL) or E. coli (107/mL) for 60 min in two different sera. EDTA at a final concentration of 20 mM should be added to serum after activation to avoid in vitro activation of residual nonactivated components. 2.5 Cross-Reactivity, Spiking, and Coefficients of Variation

The present assay was tested for cross-reactivity against human C3a (Fig. 2). Neither normal human plasma nor zymosan-activated human serum showed any reactivity in the assay, with values below the lower detection limit, comparable to the buffer blank (Fig. 2, left panel). The lower detection limit was defined by the lowest standard (i.e., 7.8 ng/mL when corrected for a 1/20 dilution). In order to control the precision of the assay, spiking of recombinant porcine C3a was performed in normal human plasma diluted 1/20, that is, corresponding to the dilution of the porcine samples and thus with the same protein concentration, but with no activity in the assay as described above. Spiking with 250 and 500 ng/mL of C3a gave a recovery of 229 (92%) and 487 (97%) ng/mL, respectively. Intra- and inter-assay coefficients of variation (CV) were calculated with samples containing different amounts of C3a as shown in Tables 1 and 2, respectively. The intra-assay CV was 4) (n ¼ 77) and SLE patients with low disease activity (SLEDAI  4) (n ¼ 88). (c) Anti-ficolin-2 titers in SLE patients with high disease activity (SLEDAI > 4) (n ¼ 77) with renal involvement (n ¼ 36) or without renal involvement (n ¼ 41). Horizontal lines in each group indicate the median values. Statistical analyses were performed by Mann–Whitney tests. (This figure is reproduced with permission from John Wiley & Sons as published by Colliard et al. [8])

4

Notes 1. The cDNAs encoding ficolin-2 (also called L-ficolin/P35) and ficolin-3 (also called H-ficolin/Hakata antigen) may be purchased from the Origene Trueclone human collection (ficolin2: SC303435; ficolin-3: SC 126138). 2. Fetal bovine serum is inactivated by heating at 56
C for 30 min. We do not freeze again heat-inactivated fetal calf serum, but keep it at 4
C in sterile 50 mL Falcon tubes.

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Fig. 3 Detection of anti-ficolin-3 autoantibodies in patients with SLE. (a) Binding of anti-ficolin-3 autoantibodies to immobilized ficolin-3. Sera from SLE patients and healthy controls were added in serial dilutions. Results represent the means  standard deviation. (b) Anti-ficolin-3 autoantibodies in serum samples. Antificolin-3 autoantibodies were measured in 48 samples from healthy controls and in 165 samples from patients with SLE. Horizontal lines in each group indicate the median values. Statistical analyses were performed by Mann–Whitney test. A absorbance, AU Arbitrary units. (Reproduced from Plawecki et al. [9])

3. EDTA is difficult to dissolve in H2O when the pH is below 7. To ensure fast dissolution, we mix 58.44 g of EDTA and 25 g of NaOH pellets in 500 mL of H2O, which yields a 0.4 M solution with a pH of approximately 7.8. 4. NaN3 is extremely toxic, and the stock solution (10% or 1.54 M) should be prepared under a ventilated hood. Alternatively, a ready-to-use solution can be purchased (0.1 M, SigmaAldrich) and used at a 15.4 mM final concentration. 5. Nunc Maxisorp plates are recommended for use in capturing immunoglobulins in ELISA. They have high protein-binding capacities. 6. TMB is sensitive to contamination from a variety of oxidizing agents. To avoid contamination, never pipette directly from the bottle. TMB may cause sensitization by skin contact. Wear gloves. 7. Ascorbic acid (vitamin C) is important for the production of ficolins, which have collagen-like domains that require ascorbic acid–dependent hydroxylation (of proline and lysine amino acid residues) for proper folding and stability.

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8. Proceed further with recombinant protein purification or store the supernatants at 80
C until use. 9. Multimeric ficolin-3 can be successfully produced in serum-free medium whereas ficolin-2 shows an oligomerization defect when expressed in CHO-K1 cells in the absence of FCS. However, ficolin-2 in FCS containing medium can be satisfactorily purified by affinity chromatography on CysNAc-Sepharose. Fractionation of FCS on CysNAc-Sepharose showed no detectable bovine ficolins. 10. Ficolins are directly attached to the plate by passive adsorption, using a carbonate–bicarbonate buffer at pH >9. It should be noticed that most but not all proteins bind tightly to the polystyrene surface of microplates in alkaline conditions. 11. It is recommended to use coated and noncoated wells in order to evaluate the specificity of antibody–protein binding. The subtraction of sera absorbance of noncoated wells allows to take into account only the specific antibody–protein binding. 12. It is important to tap conscientiously and vigorously the plate upside down on the paper towel to get rid of any remaining liquid between each step. 13. All sera are stored at 70
C until use, and repeated freeze– thaw cycles are avoided. Frozen sera should always be thawed on ice before making dilutions. 14. It should be kept in mind that incubation time and temperature affect signal generation. 15. While performing the last washing step, prepare the plate reader for absorbance reading. Set up the program for the wells to be read at 450 nm. 16. For the color development in the wells, color change in 15–30 min is desirable. It mainly depends on the coated protein concentration and autoantibodies (sera dilution). 17. The reading of the absorbance of the wells has to be done quickly after addition of the stop solution. 18. Because recombinant ficolins used for the coating step are home-made proteins, without standardization, it is recommended to optimize the conditions of ficolin–anti-ficolin autoantibody interaction. Therefore, several conditions of protein concentrations and sera dilutions have to be tested concomitantly (Fig. 4).

Chantal Dumestre-Pe´rard and Nicole M. Thielens

Absorbance (450 nm)

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Control + Control -

6 5 4 3 2 1 0

Ficolin-2 coating (µg/ml)

0

0.5

1

2

4

8

Sera dilutions at 1:100, 1:200, 1:400 Fig. 4 Optimization of anti-ficolin-2 autoantibody ELISA. Coating with several ficolin-2 concentrations (from 0 to 8 μg/mL) and sera dilutions of positive and negative controls (anti-ficolin-2 autoantibodies) (1:100, 1:200, and 1:400) were tested concomitantly. The selected concentration for the coating was 4 μg/mL and the sera dilution was 1:100

References 1. Liphaus BL, Kiss MH (2010) The role of apoptosis proteins and complement components in the etiopathogenesis of systemic lupus erythematosus. Clinics (Sao Paulo) 65:327–333. Review 2. Dumestre-Pe´rard C, Clavarino G, Colliard S, Cesbron JY, Thielens NM (2018) Antibodies targeting circulating protective molecules in lupus nephritis: interest as serological biomarkers. Autoimmun Rev 17(9):890–899. Review 3. Gatto M, Iaccarino L, Ghirardello A, Punzi L, Doria A (2016) Clinical and pathologic considerations of the qualitative and quantitative aspects of lupus nephritogenic autoantibodies: a comprehensive review. J Autoimmun 69:1–11 4. Yoshizawa S, Nagasawa K, Yae Y, Niho Y, Okochi K (1997) A thermolabile beta 2-macroglycoprotein (TMG) and the antibody against TMG in patients with systemic lupus erythematosus. Clin Chim Acta 264 (2):219–225 5. Krarup A, Thiel S, Hansen A, Fujita T, Jensenius JC (2004) L-ficolin is a patternrecognition molecule specific for acetyl groups. J Biol Chem 279 (46):47513–47519 6. Jacquet M, Lacroix M, Ancelet S, Gout E, Gaboriaud C, Thielens NM et al (2013)

Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway. J Immunol 190:3721–3731 7. Lacroix M, Dumestre-Pe´rard C, Schoehn G, Houen G, Cesbron J-Y, Arlaud GJ et al (2009) Residue Lys57 in the collagen-like region of human L-ficolin and its counterpart Lys47 in H-ficolin play a key role in the interaction with the mannan-binding lectin-associated serine proteases and the collectin receptor calreticulin. J Immunol 182:456–465 8. Colliard S, Jourde-Chiche N, Giovanna Clavarino G, Sarrot-Reynauld F, Gout E, Deroux A, Fouge`re M, Bardin N, Bouillet L, Cesbron J-Y, Thielens N, Dumestre-Pe´rard C (2018) Anti-ficolin-2 autoantibodies in SLE patients with active nephritis. Arthritis Care Res 70(8):1263–1268 9. Plawecki M, Lheritier E, Clavarino G, JourdeChiche N, Ouili S, Paul S, Gout E, SarrotReynauld F, Bardin N, Boe¨lle P-Y, Chiche L, Bouillet L, Thielens N, Cesbron J-Y, Dumestre-Pe´rard C (2016) Association between the presence of autoantibodies targeting Ficolin-3 and active nephritis in patients with systemic lupus erythematosus. PLoS One 11(9):1–15

Chapter 13 Detection of Anti-C3b Autoantibodies by ELISA Maria Radanova, Lubka T. Roumenina, and Vasil Vasilev Abstract Autoantibodies against complement proteins are involved in the pathological process of many diseases, including lupus nephritis, C3 glomerulopathies, and atypical hemolytic uremic syndrome. This method describes the detection of autoantibodies targeting the central complement component C3 by ELISA. These autoantibodies (IgG) are detected in up to 30% of the patients with lupus nephritis and more rarely in cases with C3 glomerulopathies. These autoantibodies recognize the active fragment C3b and have overt functional consequences. They enhance the formation of the C3 convertase and prevent the inactivation of C3b by Factor H and complement receptor 1. Moreover, they enhance the deposition of complement activation fragments on activator surfaces, such as apoptotic cells. The data currently available on the relations of anti-C3 autoantibodies with clinical, laboratory, and histological markers for activity of lupus nephritis, as well as the relations of anti-C3 with classical immunological markers for activity of autoimmune process in patients with lupus nephritis, such as hypocomplementemia and high levels of anti-dsDNA, could identify these autoantibodies as a potential marker for evaluation the activity of lupus nephritis. These autoantibodies correlate with the disease severity and can be used to identify patients with lupus nephritis who were prone to flare. Therefore, the detection of such autoantibodies could guide the clinicians to evaluate and predict the severity and to manage the therapy of lupus nephritis. Key words Anti-C3b autoantibodies, Anti-C3 autoantibodies, Complement, C3, ELISA, Autoantibodies, Lupus nephritis, C3 glomerulopathy

1

Introduction The autoantibodies against activated forms of C3 were discovered about 70 years ago and named immunoconglutinins [1]. Later studies showed binding to immobilized C3, which adopts a C3 (H2O)-like conformation, and to C3b, iC3b, and C3c with variable intensities but very rarely to C3d and C3a and not to fluid-phase native C3 [2–9]. These antibodies recognize epitopes, shared between C3(H2O)/C3b/iC3b/C3c; therefore, for screening anti-C3 activation fragments a C3b autoantibody is the most suitable, since it contains all recognized epitopes [10]. Anti-C3b autoantibodies have been established in patients with different autoimmune diseases [9, 11–15] including systemic lupus

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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erythematosus (SLE) [4–6, 16] and lupus nephritis (LN) [7, 8] and they have overt functional activity. It was found that anti-C3b IgG in patients with SLE, LN, or C3 glomerulopathy (C3G), and Immune Complex glomerulonephritis (IC-GN) trigger overactivation of the complement cascade by the alternative pathway [4, 7, 9, 15]. The anti-C3b also perturb the binding of the negative regulators Factor H and complement receptor 1 to C3b, causing mostly loss of regulation and to a milder degree—direct overactivation of the C3 convertase [10]. The presence of ant-C3b IgG correlated with hypocomplementemia and with high levels of anti-dsDNA [7]. In lupus nephritis patients anti-C3b are detected in up to 30% of them and have diagnostic and prognostic significance [7, 8]. These autoantibodies correlate with the disease severity and can be used to identify at risk patients for disease flare [8]. Therefore, the detection of anti-C3b could guide the clinicians to evaluate and predict the severity and to manage the therapy of lupus nephritis. The presence of anti-C3b can be detected routinely by ELISA. Here we describe in detail a method of measuring autoantibodies targeting the central complement component C3 by ELISA.

2

Materials All buffers are to be prepared with deionized water and analytical grade reagents.

2.1 Collection of the Blood Samples

1. Tubes for blood collection for plasma or serum (see Note 1). 2. Centrifuge. 3. Freezers (
80  C).

2.2

ELISA

1. Microplates for ELISA (96 wells). 2. Purified C3b (see Note 2) for diagnostic purpose or C3 fragments (C3a, C3c, iC3b, C3d, and C3) for epitope mapping (see Note 3). 3. Phosphate Buffered Saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4. PBS can be made as 10 PBS stock solution. To prepare 1 L 10 PBS dissolve the reagents: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4 in 800 mL of H2O. Adjust the pH to 7.4 with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and store them in
20  C. 4. Tween–phosphate buffered saline (TPBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, and 0,05% Tween 20. To prepare 1 L TPBS dilute 100 mL of previously made 10 PBS, pH 7.4 to 800 mL H2O add 5 mL

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of 10%Tween 20 solution, and then add H2O to 1 L. To prepare 10 mL 10%Tween 20 add 1 mL of Tween 20 (cut end of pipette tip to aspirate Tween 20 easily and pipet very slowly) to 9 mL H2O and disperse several times into the solution. 5. Serum or plasma sample, positive for anti-C3b antibodies to be used as a positive control and as a standard (see Note 4). Store at
20  C or
80  C until use. 6. Anti-human IgG peroxidase conjugated antibody (see Note 5). 7. TMB (3,30 ,5,50 -tetramethylbenzidine liquid substrate system) revealing reagent (see Note 6). Store at 2–8  C. 8. Sulfuric acid (H2SO4): 1 M solution in water. Warning: add always the acid in water and never the opposite. Get a 1 L volumetric flask and fill in with 500 mL of water. Then, slowly, added 98.079 g (or 53.3 mL) of sulfuric acid (see Note 7). 9. Microplate reader.

3

Methods Carry out all the steps of the ELISA at room temperature (see Note 8). 1. Blood collection: Collect blood and centrifuge the tubes at 2000  g for 10 min at room temperature. Divide plasma or serum into aliquots (200–300 μL) and store at
80  C until tested. Avoid repeated freezing and thawing of the sample. 2. Prepare the plan of the plate, adding two coated wells and one uncoated control well per sample. 3. Dilute C3b to a final concentration of 10 μg/mL in PBS. Prepare the final volume need to cover the planned wells with 50 μL/well. Add an excess for 4 wells, to make sure the volume will be enough to pipet correctly. 4. Coat the plate by adding 50 μL to each well of the microplate, according to the plan, using a multichannel pipette. 5. Incubate overnight at 2–8  C or 1 h at room temperature (see Note 9). 6. Remove the contents from each well by returning of the plate over a trash recipient or by pipetting and tap the plate on absorbent paper to remove the excess of the liquid. 7. Wash the plate three times with TPBS by a multichannel pipette (200 μL/well). 8. Block the plate with PBS–0.4% Tween 20 (see Note 10), dispensing 200 μL/well with a multichannel pipette.

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9. Incubate for 1 h at room temperature. 10. During the blocking time dilute the plasma (or serum) samples to be tested and the positive control 1/100 by adding 3 μL of plasma in 300 μL of TPBS. Use samples of healthy donors as a negative control and to determine the normal range (see Note 11). 11. If a standard is to be used, dilute the positive sample 1/50, 1/100, 1:200, 1:400, 1:800, 1:1600, and 1/3200, and buffer only in TPBS, in order to obtain a reference curve. 12. Remove the contents from each well by returning of the plate over a trash recipient or by pipetting and tap the plate on absorbent paper to remove the excess of the liquid. 13. Wash the plate 3 times with TPBS by a multichannel pipette (200 μL/well). 14. Add the diluted samples (50 μL/well) in duplicate and also in the uncoated control wells (see Note 12). 15. Incubate for 1 h at room temperature. 16. Dilute the anti-human IgG-HRP according to the manufacturer’s instructions or the internal optimization in TPBS (see Note 13). In our experience, we use anti-human IgG-HRP from Southern Biotech, which we dilute 1:1000. 17. Discard the content of the plate and wash the plate five times with TPBS by a multichannel pipette (200 μL/well). 18. Add 50 μL of diluted anti-human IgG-HRP in the respective wells, with a multichannel pipette. 19. Incubate for 1 h at room temperature. 20. Discard the content of the plate and wash the plate three times with TPBS by a multichannel pipette (200 μL/well). 21. Add 50 μL/well TMB solution (see Note 14). 22. Incubate for 10 min at room temperature in dark (e.g., cover the plate with aluminum foil). 23. Stop the reaction by add 50 μL/well of 1 M H2SO4. The sulfuric acid solution should be added to the wells in the same order and at the same rate as was the TMB solution. 24. Gently tap the plate on the bench top to disperse the substrate color development evenly. 25. Determine the absorbance reading at 450 nm rapidly after completing the experiment. 26. Calculate the average of the two repetitions per sample and subtract the value of the respective uncoated well. This gives the corrected OD for each sample.

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27. The normal range is calculated as an average of the corrected OD values of all healthy control samples +3 standard deviations. 28. The resulting OD of each tested sample is compared with the upper limit of the normal range to determine the positivity (see Notes 15 and 16).

4

Notes 1. Although EDTA-plasma is standardly used for this assay, using of serum sample is also possible. The normal range has to be determined for each laboratory for each type of blood product. 2. Purified C3 can be also used for this assay. Comparative analyses revealed that the patients who are positive for anti-C3 are also positive against C3b [7]. The normal range has to be standardized for the antigen used. Since most of the publications [8] used C3b, it is recommended to use C3b for easier comparison. 3. Epitope mapping revealed that the main epitopes are located in the domains composing C3c, since the antibodies recognized mainly C3b/iC3b/C3c but not/very rarely C3a and C3d [7]. 4. An international standard for quantification of anti-C3b autoantibodies is lacking. Therefore, the results are either expressed as optical density (OD) or as arbitrary units, compared to a positive sample, in-house designated as a standard. This lack of standard hampers the correct comparison of the results between laboratories and warrants further standardization of this assay. 5. If needed, anti-human IgG alkaline phosphatase conjugated antibody could be also used upon internal validation. 6. o-Phenylenediamine dihydrochloride can be used as a substrate as well, when the detection is done with peroxidase conjugate. 7. 1 M HCl can be used as well to stop the reaction. 8. We noticed that incubation at room temperature gives lower background compared to incubations at 37  C. 9. Both strategies of coating give good results. Nevertheless, when one of the two is selected, it has to be kept for the future experiments for standardization purpose. 10. An alternative blocking buffer is 1% BSA. A test for comparison between 1% BSA and PBS–0.4% Tween 20 could be performed in house to determine the condition giving lower background. 11. Determining the normal range is critical in order to determine the cutoff of positivity. Use 100 healthy control samples and

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calculate the mean + 3 standard deviations to determine an accurate cutoff. In each run patient samples are tested; several healthy donors’ samples have to be added as negative controls of the assay. 12. The control wells are crucial in the interpretation of the results. Some samples give a high signal but also a background on uncoated wells. If uncoated well is not used, these samples will be considered as positive, while the signal comes from nonspecific binding. 13. If other immunoglobulin classes are to be tested (IgM, IgA), the appropriate antibody should be selected (anti-human IgM-HRP, etc.). Anti-IgG subclass antibodies can be used as well to determine the prevalence of the subclasses (IgG1, IgG2, IgG3, and IgG4). 14. The incubation time with TMB has to be determined for each laboratory as the optimal time of obtaining strong signal from the samples but no/low signal from the uncoated control and the healthy controls. Once fixed, this time has to be respected for future experiments. 15. The positivity of a given sample has to be validated if the test is used for diagnostic purpose by repetition of the experiment. For research purpose it can be validated by IgG purification and testing the binding by surface plasmon resonance [17]. 16. In some diseases the testing of anti-C3b gave negative results, suggesting that these antibodies are not relevant for this pathology, such as the monoclonal gammopathy associatedC3 glomerulopathy [18]. References 1. Lachmann PJ (1967) Conglutinin and immunoconglutinins. Adv Immunol 6:479–527 2. Janssen BJ, Huizinga EG, Raaijmakers HC et al (2005) Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437:505–511 3. Janssen BJ, Christodoulidou A, McCarthy A et al (2006) Structure of C3b reveals conformational changes that underlie complement activity. Nature 444:213–216 4. Nilsson B, Ekdahl KN, Svarvare M et al (1990) Purification and characterization of IgG immunoconglutinins from patients with systemic lupus erythematosus: implications for a regulatory function. Clin Exp Immunol 82:262–267 5. Nilsson B, Ekdahl KN, Sjoholm A et al (1992) Detection and characterization of immunoconglutinins in patients with systemic lupus

erythematosus (SLE): serial analysis in relation to disease course. Clin Exp Immunol 90:251–255 6. Kenyon KD, Cole C, Crawford F et al (2011) IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity. J Immunol 187:2101–2111 7. Vasilev VV, Noe R, Dragon-Durey MA et al (2015) Functional characterization of autoantibodies against complement component C3 in patients with lupus nephritis. J Biol Chem 290:25343–25355 8. Birmingham DJ, Bitter JE, Ndukwe EG et al (2016) Relationship of circulating anti-C3b and anti-C1q IgG to lupus nephritis and its flare. Clin J Am Soc Nephrol 11:47–53 9. Marinozzi MC, Roumenina LT, Chauvet S et al (2017) Anti-factor B and anti-C3b

Anti-C3b Autoantibodies autoantibodies in C3 glomerulopathy and Ig-associated membranoproliferative GN. J Am Soc Nephrol 28:1603–1613 10. Vasilev VV, Radanova M, Lazarov VJ et al (2019) Autoantibodies against C3b-functional consequences and disease relevance. Front Immunol 10:64 11. Potter BJ, Brown DJ, Watson A et al (1980) Complement inhibitors and immunoconglutinins in ulcerative colitis and Crohn’s disease. Gut 21:1030–1034 12. Ngu JL, Soothill JF (1969) Immunoconglutinin and complement changes in children with acute nephritis. Clin Exp Immunol 5:557–566 13. Ngu JL, Barratt TM, Soothill JF (1970) Immunoconglutinin and complement changes in steroid sensitive relapsing nephrotic syndrome of children. Clin Exp Immunol 6:109–116 14. Ngu JL, Blackett K (1970) Complement and immunoconglutinin changes in the nephrotic syndrome of adult Africans. J Trop Med Hyg 73:250–254

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15. Chen Q, Muller D, Rudolph B et al (2011) Combined C3b and factor B autoantibodies and MPGN type II. N Engl J Med 365:2340–2342 16. Durand CG, Burge JJ (1984) A new enzymelinked immunosorbent assay (ELISA) for measuring immunoconglutinins directed against the third component of human complement. Findings in systemic lupus erythematosus. J Immunol Methods 73:57–66 17. Noe R, Chauvet S, Togarsimalemath SK et al (2019) Detection of autoantibodies to complement components by surface Plasmon resonance-based technology. Methods Mol Biol 1901:271–280 18. Chauvet S, Roumenina LT, Aucouturier P et al (2018) Both monoclonal and polyclonal immunoglobulin contingents mediate complement activation in monoclonal gammopathy associated-C3 glomerulopathy. Front Immunol 9:2260

Chapter 14 Detection of Complement Factor B Autoantibodies by ELISA Miha´ly Jo´zsi and Barbara Uzonyi Abstract Antibodies to autoantigens are implicated in a large number of diseases. Such autoantibodies may cause pathological activation of complement, an ancient humoral recognition and effector system of innate immunity; in addition, complement components or regulators may be target of autoantibodies and cause abnormal complement activation or function. Autoantibodies to complement proteins are in particular involved in kidney diseases. Those binding to complement convertase enzymes can cause enhanced stability of convertases and their increased resistance to regulation, thus promoting complement turnover. Here, we describe an ELISA method to detect factor B autoantibodies that bind to and stabilize the alternative complement pathway C3 convertase enzyme, C3bBb. Key words Autoantibody, C3 convertase, Complement, ELISA, Factor B, Nephritic factor

1

Introduction Complement is an essential component of innate immunity, participating in numerous processes such as protection against infection, disposal of cellular waste and immune complexes, and activation of various immune and nonimmune cells [1]. Autoantibodies to complement components are detected in various diseases, including systemic lupus erythematosus, atypical hemolytic uremic syndrome, and C3 glomerulopathies [2]. For some anti-complement autoantibodies a pathogenic relevance is confirmed (e.g., anti-factor H), while for others it is less clear (e.g., some so-called nephritic factors, NeFs). Antibodies against the alternative and classical complement C3 convertases (C3NeFs and C4NeFs) and the C5 convertases (C5NeFs) are thought to stabilize the respective enzymes, thus leading to their prolonged activity and, consequently, enhanced complement activation [3–6]. These autoantibodies are believed to recognize a neoepitope on the formed convertases [7]. However, their pathologic relevance is uncertain as assays are not standardized; the presence of autoantibodies is often not directly proven but inferred from functional assays (e.g., hemolytic

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Autoantibodies to the alternative pathway C3 convertase. C3 nephritic factors (C3NeFs) are autoantibodies binding to a neoepitope on the C3bBb convertase formed by both convertase components C3b and Bb, and stabilize the convertase resulting in increased life-time of the convertase and thus enhanced cleavage of C3 into C3b (left panel). Factor B autoantibodies (FB-AAs) bind to the Bb component of the convertase and have similar convertase stabilizing effect (right panel)

assays, where abnormal hemolysis may be caused by other anticomplement autoantibodies or mutant proteins), and often simply reported without functional characterization. A correlation between their levels and disease activity is in part unclear, and they are also described in healthy individuals. More recently, autoantibodies against components of the alternative pathway C3 convertase C3bBb, that is, C3b and factor B (FB), were also described and characterized [8–12]. There are few reports on FB autoantibodies in C3 glomerulopathy. The FB autoantibodies bind to the Bb part in the convertase (Fig. 1) [8, 11]. The purfied IgG fractions containing such autoantibodies were shown to stabilize the C3 convertase and result in increased C3a generation but decreased C5a generation in two cases [8, 10]. When studied in a larger patient cohort, their frequency of ~2.5% was reported in C3 glomerulopathy (3 in 118 patients) and ~39% in Ig-associated membranoproliferative glomerulonephritis (9 in 23 patients) [11]. The anti-FB IgG increased the generation of C3a and Bb but did not enhance the formation of sC5b-9 complex in normal serum in vitro [11]. Thus, FB autoantibodies are apparently rarely associated with C3G and further studies are needed to clarify their potential disease causing or disease modifying effect. Here, we describe the method used to detect FB autoantibodies in patient samples by ELISA.

2

Materials Purified factor B; phosphate-buffered saline (PBS); Tween 20; bovine serum albumin (BSA); anti-human IgG conjugated with horseradish peroxidase (HRP); 3,30 ,5,50 -tetramethyl-benzidine (TMB) solution.

Detection of anti-FB Autoantibodies

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Method 1. Coat microtiter plate wells with purified FB by adding 50μl solution of 5μg/ml FB diluted in PBS, and parallel wells with BSA (used as irrelevant antigen control) by adding 50μl solution of 5μg/ml BSA diluted in PBS, overnight at 4  C or for 2 h at 20  C. 2. Wash three times with 100μl of PBS containing 0.05% Tween 20 (PBST). 3. Block remaining binding sites with 3% BSA dissolved in PBST by adding 100μl blocking solution for 1 h at 20  C. 4. Wash three times with 100μl PBST. 5. Add samples to be tested and control samples (e.g., healthy donors’ sera or plasma), diluted 1:50 in PBST, in 50μl for 1 h at 20  C (see Notes 1–3). 6. Wash three times with 100μl PBST. 7. Add HRP-conjugated anti-human IgG (diluted 1:1000 in PBST) in 50μl for 1 h at 20  C (see Note 2). 8. Wash three times with 100μl PBST, then once with PBS. 9. Add 50μl TMB solution to each well; after development of blue color, stop the reaction by adding 25μl 2 N H2SO4 to each well. 10. Read the absorbance at 450 nm using an ELISA reader (see Notes 4 and 5). A typical result of FB autoantibody detection by ELISA is shown in Fig. 2.

Fig. 2 Detection of FB autoantibodies by ELISA. Result of a typical experiment, showing IgG binding to immobilized FB from patient’s serum but not from serum of a healthy donor. BSA is used as an irrelevant antigen (negative control)

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Notes 1. Serum or EDTA-plasma samples diluted in PBST may be analyzed for the presence of anti-FB autoantibodies. 2. The detecting HRP-conjugated anti-human IgG antibody may be replaced by subclass specific antibody to determine the isotype(s) of anti-FB autoantibodies of the patients. Similarly, HRP-conjugated anti-IgM or anti-IgA may be used to detect non-IgG immunoglobulins reacting with FB; in this case, IgG-depleted serum or plasma can be used to remove highaffinity IgGs that may prevent binding of, for example, IgM. To this end, the serum/plasma sample can be incubated with commercial Protein G-beads to remove IgG. 3. Monoclonal antibodies to the Ba and Bb fragments can be used to determine the binding site of the autoantibodies [8]. In this case, after the blocking step, the FB-coated wells are incubated without or with either mAbs, followed by the addition of the diluted patient sample. 4. Other assay formats, for example using membranes or glass slides with the target and control proteins spotted on the surface, may be adopted, as described [8]. 5. Purified IgG from the autoantibody positive patients may be used to functionally characterize the FB autoantibodies and confirm their effect on convertase stability and activity, in surface-based convertase ELISA as described [8] or surface plasmon resonance assay [13].

Acknowledgments The work of the authors is supported by project no. FIEK_16-12016-0005, implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the FIEK_16 funding scheme. References 1. Ricklin D, Hajishengallis G, Yang K, Lambris JD (2010) Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11(9):785–797. https://doi.org/ 10.1038/ni.1923 2. Trouw LA, Roos A, Daha MR (2001) Autoantibodies to complement components. Mol Immunol 38(2–3):199–206 3. Spitzer RE, Vallota EH, Forristal J, Sudora E, Stitzel A, Davis NC, West CD (1969) Serum

C’3 lytic system in patients with glomerulonephritis. Science 164(3878):436–437 4. Daha MR, Fearon DT, Austen KF (1976) C3 nephritic factor (C3NeF): stabilization of fluid phase and cell-bound alternative pathway convertase. J Immunol 116(1):1–7 5. Halbwachs L, Leveille´ M, Lesavre P, Wattel S, Leibowitch J (1980) Nephritic factor of the classical pathway of complement: immunoglobulin G autoantibody directed against the

Detection of anti-FB Autoantibodies classical pathway C3 convetase enzyme. J Clin Invest 65(6):1249–1256 6. Marinozzi MC, Chauvet S, Le Quintrec M, Mignotet M, Petitprez F, Legendre C, Cailliez M, Deschenes G, Fischbach M, Karras A, Nobili F, Pietrement C, DragonDurey MA, Fakhouri F, Roumenina LT, Fremeaux-Bacchi V (2017) C5 nephritic factors drive the biological phenotype of C3 glomerulopathies. Kidney Int 92(5):1232–1241. https://doi.org/10.1016/j.kint.2017.04.017 7. Daha MR, Van Es LA (1981) Stabilization of homologous and heterologous cell-bound amplification convertases, C3bBb, by C3 nephritic factor. Immunology 43(1):33–38 8. Strobel S, Zimmering M, Papp K, Prechl J, Jo´zsi M (2010) Anti-factor B autoantibody in dense deposit disease. Mol Immunol 47 (7–8):1476–1483. https://doi.org/10.1016/ j.molimm.2010.02.002 9. Jo´zsi M, Reuter S, Nozal P, Lo´pez-Trascasa M, Sa´nchez-Corral P, Proha´szka Z, Uzonyi B (2014) Autoantibodies to complement components in C3 glomerulopathy and atypical hemolytic uremic syndrome. Immunol Lett 160(2):163–171. https://doi.org/10.1016/j. imlet.2014.01.014 10. Chen Q, Mu¨ller D, Rudolph B, Hartmann A, Kuwertz-Bro¨king E, Wu K, Kirschfink M,

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Skerka C, Zipfel PF (2011) Combined C3b and factor B autoantibodies and MPGN type II. N Engl J Med 365(24):2340–2342. https://doi.org/10.1056/NEJMc1107484 11. Marinozzi MC, Roumenina LT, Chauvet S, Hertig A, Bertrand D, Olagne J, Frimat M, Ulinski T, Descheˆnes G, Burtey S, Delahousse M, Moulin B, Legendre C, Fre´meaux-Bacchi V, Le Quintrec M (2017) Antifactor B and anti-C3b autoantibodies in C3 Glomerulopathy and Ig-associated Membranoproliferative GN. J Am Soc Nephrol 28 (5):1603–1613. https://doi.org/10.1681/ ASN.2016030343 12. Vasilev VV, Radanova M, Lazarov VJ, DragonDurey MA, Fremeaux-Bacchi V, Roumenina LT (2019) Autoantibodies against C3b-functional consequences and disease relevance. Front Immunol 10:64. https://doi. org/10.3389/fimmu.2019.00064 13. Noe R, Chauvet S, Togarsimalemath SK, Marinozzi MC, Radanova M, Vasilev VV, Fremeaux-Bacchi V, Dragon-Durey MA, Roumenina LT (2019) Detection of autoantibodies to complement components by surface Plasmon resonance-based technology. Methods Mol Biol 1901:271–280. https://doi. org/10.1007/978-1-4939-8949-2_24

Chapter 15 Detection of C3 Nephritic Factor by Hemolytic Assay Melchior Chabannes, Ve´ronique Fre´meaux-Bacchi, and Sophie Chauvet Abstract C3 nephritic Factor (C3NeF) is autoantibody that binds neoepitopes of the C3 convertase C3bBb, resulting in a stabilization of the enzyme. First functional characterizations of C3NeF were performed by hemolytic assays using preactivated sheep erythrocytes (bearing C3b). Sheep erythrocytes are beforehand sensitized with an anti-sheep red blood cell stroma antibody produced in rabbit (hemolysin). Sensitized sheep erythrocytes will initiate cascade complement activation via the classic pathway, followed by alternative pathway amplification loop, resulting in C3b covalent binding to cell surface. Sheep erythrocytes bearing C3b permit the alternative pathway exploration, in particular decay of alternative pathway C3 convertase. Key words C3Nef, C3 Nephritic factor, C3 glomerulopathy

1

Introduction Nephritic factors (NeF) belong to a heterogeneous group of autoantibodies targeting neoepitopes of convertases of the complement system, resulting in a stabilization of the enzymes [1]. NeF have been described in human disease in particular in patients with rare kidney disease, glomerulonephritis with isolated C3 deposits or membranoproliferative glomerulonephritis (MPGN) with immune complex deposits [2, 3], rarely in other condition such as lupus nephritis [4]. Classification of these autoantibodies can be clustered according to their ability to stabilize different convertases either from the classical (C4Nef) [5, 6] or alternative pathway (C3NeF) [7] or convertase of the terminal pathway (C5NeF) [8]. Among them, C3NeF is from far the most frequent, identified in 50–70% of patient with C3 glomerulopathy [9, 10]. It was first described in 1969, based on the observation that the serum from a patient with MPGN and hypocomplementemia was able to induce C3 cleavage, when it was mixed with normal human serum [11]. The activity of C3NeF was later attributed to its capacity to bind and stabilize either cell-bound or fluid phase AP complement convertase by its

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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incorporation into C3 convertase (C3bBb) rendering the enzyme resistant to dissociation by regulators [12]. Hemolytic assays were the first tools to detect C3Nef. Nowadays, other technical approaches have been developed to detect C3Nef including binding assays (ELISA) [10] or functional assays including with measure of C3 breakdown products (Western Blot or ELISA) [10–13] in presence of Ig from patients with C3NeF. This method chapter describes our experience of detection of C3NeF using hemolytic assays adapted from the historical methods.

2

Materials

2.1 A-Step A: Preparation of Sheep Erythrocytes

1. Cooling centrifuge high capacity type KR 4.22.

2.1.1 Common Supplies

3. All instruments used during the experience should be rinsed with distilled water and be plastic.

2.1.2 Buffers

2. Spectrophotometer allowing for reading at 541 nm wavelength—50 mL polypropylene conical tubes.

All buffers should be prepared using deionized or double distilled water and analytical grade reagents. 1. Veronal buffer saline (VBS) 5
: mix 170 g NaCl + 7.5 g sodium diethylbarbital and dissolve on ice in 2 L of water. Separately mix 11.5 g barbital in 1 L water on a hot plate (350  C). When colder, add 1 L of distilled water. Mix the two solutions. When the solution is cold, adjust the pH between 7.2 and 7.3 and adjust to a final volume of 2 L. Store at 4  C for 1 month. 2. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) 0.086 M: Mix 3.201 g EDTA with 100 mL of water. Dissolve by heating and stirring. Adjust pH to 7.5 by adding NaOH. 3. CaCl2 0.03 M: Mix 2.2 g of CaCl2+ with 500 mL of distilled water. 4. 0.1 M MgCl2: Dissolve 0.952 g MgCl2 in 100 mL of distilled water. Keep for 1 month at 4  C. 5. VBS++: (500 mL): in 500 mL VBS 1
add 2.5 mL CaCl2 0.03 M and 2.5 MgCl2 0.1 M. 6. VBS-EDTA 0.01 M: (100 mL): in 88.5 mL VBS 1
add 11.5 mL EDTA 0.086 M.

Detection of C3 Nephritic Factor by Hemolytic Assay 2.1.3 Biological Components

1. Sheep blood.

2.2 B-Step B: Preparation of Sensitized Sheep Erythrocytes

1. Cooling centrifuge high capacity type KR 4.22.

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2. All instrument used during the experience should be rinsed with distilled water and be plastic.

2.2.1 Common Supplies 2.2.2 Buffers

Preparation of buffers needed for step B is the same as for buffers in step A. 1. Veronal Buffer Saline (VBS) 5
. 2. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) 0.086 M. 3. VBS++: (500 mL): in 500 mL VBS 1
add 2.5 mL CaCl2 0.03 M and 2.5 MgCl2 0.1 M. 4. VBS-EDTA 0.01 M: (100 mL): in 88.5 mL VBS 1
add 11.5 mL EDTA 0.086 M. 5. VBS EDTA 0.04 M: In 400 mL VBS 1
add 348 mL EDTA 0.086 M.

2.2.3 Biological Components

1. Sheep erythrocytes prepared in step A. 2. Hemolysin: Anti-Sheep Red Blood Cell Stroma antibody produced in rabbit (Sigma S-1389). 3. Sera complement of guinea pig: Sigma S-163.

2.3 Step C-Preparation of Sheep Erythrocytes Bearing C3b

1. Cooling centrifuge high capacity type KR 4.22. 2. Shaking water baths 37  C. 3. Spectrophotometer wavelength.

allowing

for

reading

at

414

nm

2.3.1 Common Supplies 2.3.2 Buffers

All buffers should be prepared using deionized or double distilled water and analytical grade reagents. 1. 10% gelatin: mix 1 g of gelatin with 10 mL water. 2. 5% dextrose: mix 50 g dextrose in 1 L water. 3. GVB (750 mL): mix extemporaneously 150 mL of VBS 5
with 7, 5 mL 10% gelatin and complete with distilled water. 4. GVB-EDTA (1122 mL): mix 600 mL GVB with 522 mL EDTA 0.086 M. It must be stored at 4  C and cold when use in the experience.

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5. DGVB++(303 mL): mix 150 GVB with 150 mL 5% dextrose, then add 1.5 mL Ca2+ 0.03 M and 1.5 mL Mg2+ 0.1 M. 6. NaCl: 0.15 M: mix 8.766 g NaCl in 1 L distilled water. 2.3.3 Biological Components

1. 50 mL of sensitized sheep erythrocytes (prepared in Step B). 2. Human fresh serum. 3. Suramin (EMD, reference: 574625-200MG): prepare 640 μL at the concentration of 30 mg/mL in DGVB++ buffer. 4. Zymozan (Sigma, reference: Z4250-250MG) 2 mg/mL: boil Zymozan in NaCl 0.15 (volume equivalent to fresh human serum) for 30 min and wash (Centrifuge the solution for 5 min at 1400
g, remove the supernatant, and wash 3–4 times with NaCl 0.15 M.). Resuspend the Zymosan solution in a volume equivalent to the volume of fresh human serum volume). 5. Zymozan Activated Human Serum (ZAHS): For SAHZ preparation, see below.

2.4 Step D: Purification of Total IgG from Patient Plasma (See Note 1)

1. Phosphate-buffered saline (PBS) 1
: five liters of phosphatebuffered saline (PBS) is needed for the washing of the Protein G beads (final concentration of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl). 2. Protein G beads (GE Healthcare). 3. 0.1 M glycine hydrochloride, pH 2.8. 4. Columns (volume 1.5 mL) (e.g., Biospin chromatography column). 5. Elution buffer is 0.1 M glycine hydrochloride, pH 2.8. Weigh 11.1 g glycine hydrochloride and dissolve with 800 mL water. Adjust pH to 2.7 and bring volume to 1 L with water. 6. For the neutralization of the pH of the purified IgG, the buffer is 1.5 M Tris, pH 8. To prepare 100 mL, weigh out 18,165 mg Tris and add to 80 mL of water on a magnetic stirring plate to mix the solution and adjust the pH to 8 with HCl. Adjust the final volume to 100 mL.

2.5 Step E-C3Nef Assays with Sheep Erythrocytes Bearing C3b

1. Cooling centrifuge high capacity type KR 4.22. 2. Shaking water baths 37  C. 3. Spectrophotometer allowing for reading at 414 nm wavelength. Hemolysis tube.

2.5.1 Common Supplies 2.5.2 Buffers

All buffers should be prepared using deionized or double distilled water and analytical grade reagents. 1. GVB (100 mL): mix extemporaneously 20 mL of VBS 5
with 1 mL 10% gelatin and complete with distilled water to a final volume of 100 mL.

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2. GVB-EDTA 0.04 M (93.5 mL): mix 50 mL GVB with 43.5 mL EDTA 0.086 M. 3. DGVB++(100 mL): mix 50 GVB with 50 mL 5% dextrose, then add 0.5 mL Ca2+ 0.03 M and 0.5 mL Mg2+ 0.1 M. 2.5.3 Biological Components

1. Sheep erythrocytes bearing C3b (prepared in step C). 2. Rat serum 1/20 diluted in GVB-EDTA 0.04 M buffer (1 mL of rat serum in 19 mL of GVB-EDTA 0.04). 3. Purified complement factor B (1 mg/mL). 4. Purified complement factor D (100 μg/mL). 5. Total Purified IgG (prepared in step D).

3

Methods

3.1 A-Step A: Preparation of Sheep Erythrocytes

1. Take two falcons of almost 30 mL of sheep blood, preagitated for 30 min. 2. Centrifuge for 5 min, 1400
g at 4  C. Remove the supernatant comprising plasma and white blood cells. 3. It is possible to test the buffer during this step. Take 100 μL of sheep blood in 2.9 mL of each buffer in separate tubes. Centrifuge for 5 min, 1400
g at 4  C. If the buffers are good, no hemolysis will be observed. 4. Wash the sheep erythrocytes twice with 30 mL of VBS-EDTA 0.01 M. 5. Centrifuge for 5 min 1400
g, at 4  C. Remove the supernatant, then wash them twice in 30 mL VBS++. Remove the supernatant after the last washing. 6. Then adjust cell suspension at 109 cells/mL in VBS++ (see Note 2). First in a clean, dry beaker resuspend the SE in the average VBS++ volume calculated. Then adjust the cell suspension at 109 cells/mL, take 200 μL of the cells and dilute them in 2.8 mL of distilled water. Measure the OD of the solution with the spectrophotometer at 515 nm and calculate the suitable dilution of the cells needed to obtain an OD equal to 0.700, according to the equation: 7. Final volume ¼ ðOD sample at A515 =0:700Þ
initial volume 8. Verify the adjustment by repeating the previous step to obtain the good OD515 (¼0.700).

3.2 B-Step B: Preparation of Sensitized Sheep Erythrocytes

1. In the adjusted cell suspension, add the equivalent volume of VBS++ then the hemolysin at the dilution predetermined (see Table 1 for the determination of hemolysin dilution and see Note 3 for an example). 2. Gently stir manually, then incubate for 10 min at room temperature.

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Table 1 Test for determination of the appropriate dilution of hemolysin T0 T100 T Hemolysin diluted / in VBS++ (mL)

/

/

1/600 1/800 1/1000 1/1200 1/1400 1/1600 1/1800 1/2000 0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5 0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Cells 109 (mL)

0.5 0.5

Incubation

10 min at room temperature

VBS++ (mL)

6

/

7

5.5

5.5

5.5

5.5

5.5

5.5

5.5

5.5

Sera complement diluted 1/20 VBS++ (mL)

1

1

/

1

1

1

1

1

1

1

1

Distilled water (mL)

/

6

/

/

/

/

/

/

/

/

/

Incubation

45 min at 37  C Centrifuge for 5 min 4  C 1400
g

Reading

Spectrophotometer at 515 nm

3. Dispense in polypropylene conical tubes (almost 30 mL per tube). 4. Centrifuge for 5 min, 1400
g at 4  C. Remove the supernatant and wash them twice with VBS-EDTA 0.004 M. 5. Resuspend the sheep erythrocytes in VBS-EDTA 0.004 M at the same volume previously determined for 109 cell/mL. 6. Aliquot in 50 mL falcon, (5 mL by falcon), and store at 4  C. 3.3 C-Step C: Preparation of Sheep Erythrocytes Bearing C3b

1. Take fresh human serum and measure the volume.

3.3.1 Preparation of Zymozan-Activated Human Serum (ZAHS) Solution

3. Centrifuge the solution for 5 min at 1400
g, remove the supernatant, and wash 3–4 times with NaCl 0.15 M.

2. Prepare an equivalent volume of Zymosan at the concentration of 2 mg/mL in NaCl 0.15 M. Boil the solution at 150–200  C for 30 min in a closed Erlenmeyer.

4. Resuspend the solution in the original volume (equivalent of the fresh human serum volume). 5. Then add the fresh human serum. 6. Incubate for 45 min, at 37  C in a shaking water bath. 7. Centrifuge for 5 min at 1400
g. 8. Keep the supernatant corresponding to ZAHS. 9. Test the CH50 activity of the obtained ZASH. It must be slightly positive (i.e., 10–15%). 10. Store aliquot of 3.2 mL at 80  C.

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3.4 Preparation of Sheep Erythrocytes Bearing C3b

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First prepare the ½ diluted ZASH and Suramin as indicated above. Be sure that the DGVB-EDTA buffer is ice-cold. 1. Take 20 mL of sensitized sheep erythrocytes (109 cell/mL) (prepared in step B), centrifuge them for 5 min at 1400
g, remove the supernatant, and wash them twice with DGVB++. 2. Then adjust cell suspension to 2
108 cells/mL in DGVB++ (see Note 4). 3. First, resuspend the sensitize SE in the estimated volume of DGVB++ in a clean, dry Erlenmeyer. 4. Then adjust the cell suspension to 2
108 cells/mL, take 50 μL of the cells and dilute them in 2.95 mL of distilled water. Measure the OD of the solution with the spectrophotometer at 414 nm and calculate the adapted dilution of the cells needed to obtain an OD equal to 0.286, according to the equation: Final volume ¼ ðOD sample at A414 =0:286Þ
initial volume 5. In 80 mL of adjusted SE, add the diluted ZASH and stir strongly manually for 1 min at 37  C, in a water bath. 6. Add 640 μL of suramin (30 mg/mL) and stir strongly manually during 2 min at 37  C in water bath. 7. Stop the reaction by adding 240 mL of ice-cold GVB-EDTA 0.04 M. 8. Dispense the solution in 8 conical tubes, centrifuge for 5 min, 1400
g at 4  C, remove the supernatant, and wash them twice with 30 mL of ice-cold GVB-EDTA 0.04 M. 9. In a plastic clean and dry breaker, resuspend the SE in 160 mL of ice-cold GVB-EDTA. Dispense them into 8 conical tubes, then incubate for 90 min at 37  C in a shaking water bath. 10. Pooled into four conical tubes, centrifuge for 5 min, 1400
g at 4  C, remove the supernatant and wash them twice with 30 mL of ice-cold GVB-EDTA. 11. Resuspend the cell in GVB-EDTA 0.04 M at the volume previously determined in order to have a concentration of 2
108 cells/mL. 12. Aliquot by 10 mL and store at 4  C. for 4 weeks. 13. Wait for at least 3 days before utilization.

3.5 Step D: Purification of Total IgG from Patient Plasma

1. Take the needed amount of Protein G beads and transfer to a tube. The needed raw beads volume is 500 μL per sample. 2. Wash three times in PBS with slow centrifuging in between (5 min at 50
g) to remove the ethanol and its traces used for the storage of beads.

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3. Divide the beads in individual tubes, sediment by centrifugation and aspire the supernatant. 4. Add 500 μL of plasma on sedimented beads (adjust the volume of plasma with the amount of beads to scale up if necessary). 5. Incubate for 1 h on roller (Rotatory shaker) at 4  C in cold room to obtain an efficient binding of IgG on the beads. 6. Transfer the tube content to small column and wash with 10 mL of PBS minimum. 7. Check the O.D. at 280 nm of last flow through before elution to confirm the efficiency of the wash, the O.D. needs to be null for a high purity. 8. Add 10 μL of Tris 1.5 M pH 8 in a 1.5 mL tube for each 500 μL fraction to be collected in the next step to assure immediate neutralization of the IgG-containing solution after the elution step. 9. Elute the IgG by repeated additions of 4
500 μL glycine hydrochloride. Collect the fraction in 1.5 mL tubes prepared with neutralizing solution. 10. Check the OD after the fourth fraction and continue to elute until the OD is null. 11. As soon as the elution is over, wash the column in PBS (5 mL). If the beads are to be used again, add azide for longer-term storage. Keep at 4  C. 12. Pool the highest OD and measure the OD at 280 nm again to estimate the protein concentration. 13. Place the samples to be dialyzed in dialysis cassettes or tubes with appropriate volume. 14. Dialyze in a 200-fold greater volume of PBS, compared to the total sample volume, for 1 h or overnight at 4  C with magnetic stirring. Change the dialysis buffer 2 more times and dialyze for minimum 1 h. 15. Check O.D. after dialysis to make sure to not lose the protein at this step. Freeze at 80  C until use. 3.6 Step E: C3Nef Assays with Sheep Erythrocytes Bearing C3b

1. Prepare seven tubes with 100 μL of DGVB++ in tube 2–7 as indicated in figure below.

3.6.1 Determine Concentration of Factor B (FB) to Obtain 1.5 Lytic Sites per Cell (Z ¼ 1.5) (Fig. 1)

3. From FB dilution in tube 2, prepare a serial dilution of FB in tube 2–7.

2. In tube 1 and 2, add purified FB 1/1000 diluted in DGVB++ as indicated in figure below.

4. Prepare two control tubes: T0 and T100. 5. In each tube, add 100 μL of sensitized sheep erythrocytes (108 cell/mL) in DGVB++ mixed with purified FD (1 μg/mL of cell) and incubate 30 min at 30  C.

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Fig. 1 Determine concentration of FB to obtain 1.5 lytic sites per cell (Z ¼ 1.5)

6. Add 300 μL of rat serum 1/20 diluted in GVB-EDTA in each tube 1–7 and in T0 and T100 and incubate for 30 min at 37  C. 7. Stop reaction with 1.6 mL of 150 mM sodium chloride buffer in all tubes except in tube T100 where reaction is stopped by adding 1.6 mL of water (leading to osmotic complete lysis of cells). 8. Read ODtubes at 414 nm. (a) Calculation of Z. 9. Z ¼ log (1  (ODtubes  OD T0/OD T100-OD/T0) and determine concentration of FB need to obtain a Z of 1.5 for 1 mL of cells. 3.6.2 C3NeF Assay (Fig. 2)

3.7 Convertase Formation on Sheep Erythrocytes

1. In a tube, mix the volume of sheep erythrocytes bearing C3b (108 cells/mL) needed for the number of IgG samples to be tested (patients and IgG from healthy donor, see Note 5) you want to test (100 μL/tubes) with FD (1 μg/mL of cells) and adapted concentration of FB (per mL of cells) determined in previous step. 2. Prepare three control tubes. 3. T0: 100 μL of sheep erythrocytes bearing C3b (108 cells/mL) with FD (1 μg/mL of cells) without FB.

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Convertase formation

Convertase dissociation

Lysis

Purified IgG

FB+FD

100mL of cell

Rat serum

100mL of cell

100mL of cell

Fig. 2 Experimental design of the C3Nef assay

4. T100: 100 μL of sheep erythrocytes bearing C3b (108 cells/ mL) with FD (1 μg/mL of cells) without FB. 5. Three tubes Tc dissociation step).

(formation

of

convertase

without

6. Incubate for 30 min at 30  C under checking. 7. Negative control: Tb (formation of convertase followed by dissociation convertase in presence of GVB-EDTA buffer alone). 3.7.1 Convertase Dissociation

1. During convertase formation, prepare 400 μg of IgGs from patients and healthy donor in GVB EDTA for a final volume 100 μL. 2. After convertase formation, transfer IgGs from patients in corresponding tubes and incubate for 20 min at 30  C. (a) Remember that there is no dissociation step in control tube Tc: after formation of convertase, directly add 300 mL of rat serum 1/20 diluted in GVB-EDTA buffer and incubate for 30 min at 37  C and then stop the reaction with 1.6 mL of 0.15 M NaCl buffer.

3.7.2 Lysis

1. Add 300 μL of rat serum 1/20 diluted in GVB-EDTA buffer and incubate for 30 min at 37  C (with agitation). 2. After 30 min, stop the reaction with 1.6 mL of 0.15 M NaCl buffer. 3. In control tubes T0, stop the reaction with 1.6 mL of 0.15 M NaCl buffer. In the control tube T100, 1.6 mL of distillated water (to obtain osmotic lysis of all erythrocytes). 4. Read OD of each tubes at 414 nm.

Detection of C3 Nephritic Factor by Hemolytic Assay 3.7.3 Calculation of the Percentage of Stabilization

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With OD value of T0, T100 and mean value of the 3 Tc, calculate. 1. ZTc¼ LN (1  (ODmean Tc-T0)/(T100-T0)). 2. ZIgG¼ LN (1  ((ODIg-T0)/(T100-T0). 3. Percentage of stabilization ¼ ZIgG/ZTc
100.

4

Notes 1. EDTA plasma or serum can be obtained by venipuncture. After centrifugation and aliquoting, it should be stored at 80  C until utilization. 2. In our experience, 1 mL of red blood cell pellet gently mixed in 16 mL of VBS++ allows to obtain the wanted final concentration. 3. Test for determination of the right dilution of hemolysin. In the example below the right dilution is the last dilution for which the OD is 0.700. Example: Dilution

1/600

1/800

1/1000

1/1200

1/1400

1/1600

DO

700

700

700

581

540

502

Therefore, the right dilution is 1/1000. 4. Based on our experience we determined that for 20 mL of sensitized SE, the final volume will be close to 80 mL. 5. Test sufficient number of healthy donors IgG in order to determine the normal range of the assay, needed to determine if a patient is positive. References 1. Corvillo F, Okro´j M, Nozal P, Melgosa M, Sa´nchez-Corral P, Lo´pez-Trascasa M (2019) Nephritic factors: an overview of classification, diagnostic tools and clinical associations. Front Immunol 10:886 2. Servais A, Noe¨l LH, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey MA, Macher MA, Zuber J, Karras A, Provot F, Moulin B, Gru¨nfeld JP, Niaudet P, Lesavre P, Fre´meaux-Bacchi V (2012) Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 82(4):454–456 3. Smith RJH, Appel GB, Blom AM, Cook HT, D’Agati VD, Fakhouri F, Fremeaux-Bacchi V, Jo´zsi M, Kavanagh D, Lambris JD, Noris M, Pickering MC, Remuzzi G, de Co´rdoba SR,

Sethi S, Van der Vlag J, Zipfel PF, Nester CM (2019) C3 glomerulopathy - understanding a rare complement-driven renal disease. Nat Rev Nephrol 15(3):129–143 4. Walport MJ, Davies KA, Botto M, Naughton MA, Isenberg DA, Biasi D et al (1994) C3 nephritic factor and SLE: report of four cases and review of the literature. QJM 87:609–615 5. Halbwachs L, Leveille´ M, Lesavre P, Wattel S, Leibowitch J (1980) Nephritic factor of the classical pathway of complement: immunoglobulin G autoantibody directed against the classical pathway C3 convetase enzyme. J Clin Invest 65:1249–1256. https://doi.org/10. 1172/JCI109787 6. Zhang Y, Meyer NC, Fervenza FC, Lau W, Keenan A, Cara-Fuentes G et al (2017) C4

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nephritic factors in C3 glomerulopathy: a case series. Am J Kidney Dis 70:834–843. https:// doi.org/10.1053/j.ajkd.2017.07.004 7. Daha MR, Austen KF, Fearon DT (1977) The incorporation of C3 nephritic factor (C3NeF) into a stabilized C3 convertase, C3bBb (C3NeF), and its release after decay of convertase function. J Immunol 119:812–817 8. Marinozzi M-C, Chauvet S, Le Quintrec M, Mignotet M, Petitprez F, Legendre C et al (2017) C5 nephritic factors drive the biological phenotype of C3 glomerulopathies. Kidney Int 92:1232–1241 9. Servais A, Noe¨l L-H, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey M-A et al (2012) Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 82:454–464 10. Zhang Y, Meyer NC, Wang K, Nishimura C, Frees K, Jones M et al (2012) Causes of

alternative pathway dysregulation in dense deposit disease. Clin J Am Soc Nephrol 7:265–274 11. Daha MR, Fearon DT, Austen KF (1976) C3 nephritic factor (C3NeF): stabilization of fluid phase and cell-bound alternative pathway convertase. J Immunol 116(1):1–9 12. Paixa˜o-Cavalcante D, Lo´pez-Trascasa M, Skattum L, Giclas PC, Goodship TH, de Co´rdoba SR, Truedsson L, Morgan BP, Harris CL (2012) Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation. Kidney Int 82 (10):1084–1092 13. Donadelli R, Pulieri P, Piras R, Iatropoulos P, Valoti E, Benigni A et al (2018) Unraveling the molecular mechanisms underlying complement dysregulation by nephritic factors in C3G and IC-MPGN. Front Immunol 9:2329. https://doi.org/10.3389/fimmu.2018. 02329

Chapter 16 Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA Jesu´s Garcı´a-Ferna´ndez, Susana Vilches-Arroyo, Leticia Olavarrieta, Julia´n Pe´rez-Pe´rez, and Santiago Rodrı´guez de Co´rdoba Abstract The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster. Key words RCA cluster, CFH, CFHR1-5, MLPA , Next-generation sequencing (NGS), CNVs, Structural variations

1

Introduction The Regulators of Complement Activation (RCA) gene cluster (Fig. 1) spans 12 Mb of DNA and includes 16 complement genes. All the complement genes are in tandem within two gene groups, a telomeric 707 kb-long DNA segment which contains the C4BPB, C4BPA, C4BPAL1, C4BPAL2, DAF(CD55), CR2 (CD21), CR1(CD35), MCPL1, CR1L1, and MCP(CD46) genes and a centromeric 358 kb-long DNA segment that contains CFH,

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 The human RCA gene cluster in chromosome 1q31-q32. Central part of the figure shows the organization in tandem of the complement genes in two groupings separated by 10.3 Mb of DNA. Arrows are used to illustrate 50 to 30 orientation for each of the genes. Lower part depicts the CFH/CFHR1–5 gene subregion of the RCA gene cluster. Arrows represent the genes with their names above. The vertical lines under the arrows indicate the exon structure of the genes. The boxes localize the segmental duplications in this genomic region. The same letter (i.e., A, A0 , A00 ) and color-code are used to identify the different duplications

CFHR1, CFHR2, CFHR3, CFHR4, and CFHR5 [1]. These two gene groups are separated by 10.3 Mb of DNA that contains genes that are not complement related and that have very diverse functions. Both telomeric and centromeric groupings are very polymorphic in humans with hundreds of variants described for each of the complement genes. Most of these gene variants are very rare (MAF < 0.1%) and involve differences in the DNA nucleotide sequence that are readily identified by all DNA sequencing techniques. Gene variants in the RCA gene cluster also involve changes in chromosome structure, but the current databases do not have a proper representation of these genetic variants. This is because the currently used massive parallel DNA sequencing approaches do not easily identify all these structural variations. A common change in chromosome structure is the copy number variation (CNV), a type of structural variant involving alterations in the number of copies of specific regions of DNA, which can either be deleted or duplicated. The gene rearrangements that normally associate with the CNVs

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often result in the generation of novel genes, carrying internal duplications or deletions, and in hybrid genes resulting from the fusion or exchange of genetic material between two different genes. Like other types of genetic variations, some structural variants are inherited, whereas others spontaneously arise de novo. CNVs have been described at the CR1 and the CFH/CFHR1-5 gene regions of the RCA gene cluster and, in both regions, the structural variants show important associations with disease [2–9]. CNVs can be identified by different techniques like comparative genomic hybridization (CGH) arrays, multiplex ligation-dependent probe amplification (MLPA), emulsion PCR (EmPCR), or high resolution melting PCR, and also by bioinformatics analyses using public domain and commercial software, like NextGENe and ONCOCNV, designed to detect CNVs from NGS data. 1.1 CNVs in the CR1 Gene

CR1 encodes the complement receptor 1 (CD35), a membraneassociated glycoprotein expressed in many cell types but especially in erythrocytes. CR1 is a receptor for C3b and C4b that regulates complement activation and promotes opsonophagocytosis of immune complexes, cellular debris and the amyloid-beta (Aβ) peptide in Alzheimer’s disease (AD) [10, 11]. Several studies have shown that CR1 present a structural polymorphism with four CR1 alleles of different molecular sizes [12, 13]. The extracellular domain of the CR1 protein is composed of a series of repeating units, called short consensus repeats (SCRs) arranged in tandem groups of seven, known as long homologous repeats (LHRs). The most frequent CR1 allele CR1*1 (also A and F) contains four LHRs, designated as LHR-A, -B, -C, and -D. The second most common allele CR1*2 (also B and S), has a duplicated LHR-B and is composed five LHRs, while the rare CR1*3 (also C and F‘) and CR1*4 (also D) alleles, having no LHR-B and three LHR-B copies, are composed of 3 and 6 LHRs, respectively (Fig. 2). The CR1*2 allele is associated with increased risk to Alzheimer’s disease (AD). Recently, a specific high-resolution melting PCR has been developed to genotype the structural CR1 alleles [14, 15].

1.2 CNVs in the CFH and CFHR1-5 Genes

CFH encodes factor H (FH) a relatively abundant plasma glycoprotein that plays a major role in the regulation of the complement alternative pathway in plasma and in the host surfaces [16]. The CFHR1-5 genes are evolutionary related to the CFH gene. They encode FHR-1, FHR-2, FHR-3, FHR-4, and FHR-5, five plasma glycoproteins present in different concentrations ranging from 1 to 100μg/mL. Currently, the FHRs are thought to be complement deregulators that promote complement activation by competing the binding of FH to the host surfaces [17]. Sequence analyses of the CFH/CFHR1-5 gene region illustrate the existence of a number of large segmental duplications including different exons of the CFH and CFHR1-5 genes. These duplications range in size from

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Fig. 2 CNVs at the CR1 locus. The structural variation that characterizes the four CR1 alleles is related to a CNV of the LHR-B domain

1.2 to 38 kb and present a pairwise nucleotide identity from 85% to 97% ([18]; Fig. 1). They are a reminiscence of the different gene duplication events in this region that resulted in the generation of the CFH/CFHRs gene family. Because of the large segmental duplications this genomic region is prone to generate CNVs through nonhomologous recombination and gene conversion events (Table 1). Notably, gene rearrangements involving the CFH and CFHR1-5 genes are associated with a number of common and rare diseases (Table 1). MLPA is the main methodology currently used to identify CNVs in the CFH/CFHR1-5 region of the RCA gene cluster. In addition, computational tools like NextGENe and ONCOCNV are routinely used to identify CNVs in the data generated from the next generation sequencing (NGS) gene panels that today are widely used for the screening of complement gene variants. 1.3 MLPA and CNV Analysis from NGS Data

MLPA is a multiplex PCR method that has been developed commercially by MRC-Holland to detect abnormal copy numbers in a number of different regions of the human genome. This technique is easy to use and only requires a thermocycler and a capillary electrophoresis equipment (Fig. 3). The basics of the MLPA reaction is the ligation of a series of adjacent primers pairs, each pair hybridizing to a different target sequence (Fig. 3a), and the subsequent amplification of all the ligated primers pairs (the so-called probemix) using a single pair of PCR primers (Fig. 3b). The resulting amplification products are analyzed by capillary electrophoresis and the comparison of the peaks obtained with a

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Table 1 Gene rearrangements associated with the CFH/CFHR1-5 genomic region Copy number abnormality

Consequences Relevant Gene rearrangements Disease association

Prevalence

One copy CFHR3 ex1-6 One copy CFHR1 ex1-6

ΔCFHR3-CFHR1

AMD, IgAN, SLE, autoimmune aHUS [7, 8, 19–24]

Very common

One copy CFHR1 ex1-6 One copy CFHR4 ex1-10

ΔCFHR1-CFHR4

Autoimmune aHUS [21]

Several cases

Three copies CFHR3 DupCFHR3-CFHR1 C3G? ex1-6 Three copies CFHR1 ex1-6

Several cases

Three copies CFHR1 DupCFHR1-CFHR4 C3G? ex1-6 Three copies CFHR4 ex1-10

Rare

ΔCFHR3-CFHR1 CFH::CFHR1 hybrid gene

aHUS [3]

Several unrelated cases described

Three copies of CFH::CFHR1 hybrid CFHR1 ex6 gene One copy CFH ex23

aHUS [3]

Several unrelated cases described

One copy CFH ex6 One copy CFHR3 ex1-6 One copy CFHR1 ex1-5

DupCFHR3-CFHR1 aHUS [4, 6, 25] Three copies CFH CFHR1::CFH hybrid ex23 gene Three copies CFHR3 ex1-6 Three copies CFHR1 ex1-5

Several unrelated cases described

Three copies CFH ex23 One copy CFHR1 ex6

CFHR1::CFH hybrid gene

aHUS [4, 6, 25]

Several unrelated cases described

One copy CFHR3 ex5 One copy CFHR1 ex1-6 One copy CFHR4 ex1-9

ΔCFHR1-CFHR4 CFHR3::CFHR4 hybrid gene

aHUS, C3G?

Several cases

One copy CFH ex6

CFH::CFHR3 hybrid gene

aHUS [5]

Very rare (continued)

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Table 1 (continued) Copy number abnormality

Consequences Relevant Gene rearrangements Disease association

Prevalence

Three copies CFHR1ex2-4

CFHR1dupex1-4

C3G (DDD) [9]

Very rare

One copy CFHR2 ex4,5

CFHR2::CFHR5 hybrid gene

C3G (DDD) [26]

Very rare

One copy CFHR3 ex4-6

CFHR3::CFHR1 hybrid gene

C3G (C3-GN) [27]

Very rare

Three copies CFHR3 CFHR5::CFHR2 hybrid gene ex2-5 Three copies CFHR5 ex1-3

C3G (C3-GN) [28]

Very rare

Three copies CFHR5ex2-4

DupCFHR5

C3G (C3-GN) [29, 30]

Several related cases described 1 unrelated

One copy CFHR1 ex5,6 One copy CFHR4 ex1-10 One copy CFHR2 ex1-5

CFHR1::CFHR5 hybrid gene

C3G (DDD/C3-GN) [31]

Very rare

reference sample indicates the copy numbers of the target sequences (Fig. 3c). NGS is a reliable, economical and fast method to analyze both nucleotide variations and CNVs. There are multiple NGS strategies and computational tools that can address the analysis of CNVs. Those, described here, have been selected based on coverage (depth of reading), uniformity of sequencing, alignment on the target and capacity of CNV calling. For the generation of the sequence data we used a specific gene panel. Among the multiple available computational tools we have selected NextGENe and ONCOCNV. In this review, we will describe in detail these procedures.

2 2.1

Materials MLPA

Commercially available MLPA Kits from MRC-Holland include one for the FH/FHRs region of the RCA gene cluster (SALSA MLPA kit P236-A3 ARMD probemix). Additionally, the MRC-Holland Website provide the tools to develop customized sets of primers to target sequences that are not covered by their currently available MLPA Kits (see Note 1).

Detection of Genetic Rearrangements in the RCA Cluster

Fig. 3 Schematic representation of the MLPA analysis

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1. SALSA MLPA kit P236-A3 ARMD from MRC Holland (Netherlands), including: (a) SALSA MLPA Buffer. (b) SALSA Ligase-65 Enzyme. (c) Ligase buffer A. (d) Ligase buffer B. (e) SALSA PCR Primer mix. (f) SALSA Polymerase. (g) SALSA MLPA Probemix. 2. LIZ GeneScan 500 (or ROX GeneScan 500) Size Standard. 3. TE Buffer: 10 mM Tris–HCl, pH 8.2, 0.1 mM EDTA. 4. Hi-Di Formamide. 5. POP-7 polymer for capillary electrophoresis system. 6. Thermal cycler with heated lid. Periodic calibration of this equipment is strongly recommended. 7. Capillary electrophoresis system (i.e., ABI 3730 or ABI 3730XL DNA Analyzer). 8. Genemapper (ABI) software (v.4.0). 9. Coffalyser software (v.140721.1958). 2.2 CNV Analysis from NGS Data

1. Any Windows® 64-bit operating system from Vista through Windows 10, or Windows based server 2008R2 and forward.

2.2.1 NextGENe Software

2. NextGENe software (v.2.4.2).

2.2.2 ONCOCNV Software

1. LINUX operating system. 2. Perl and R installed. 3. SAMtools (http://samtools.sourceforge.net/) installed. 4. BEDTools installed.

(http://bedtools.readthedocs.org/en/latest/)

5. The following R libraries should be installed: MASS, mclust, PSCBS, DNAcopy, R.cache, scales, cwhmisc, fastICA, cghseg, and digest. 6. The fasta sequence (one file, unzipped; e.g., “hg19.fa”). 7. You need to have your data aligned (.bam files). 8. You need to have at least three control files to construct a reliable baseline. However, ONCOCNV will run with only two controls starting from version 5.4 and with just one control starting from version 5.7. Yet we recommend having at least three controls for good performance of the algorithm. 9. Download ONCOCNV.zip (or ONCOCNV.vX.X.zip).

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3 3.1

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Methods MLPA Analysis

3.1.1 Preparation of DNA

3.1.2 MLPA Assay DNA Denaturation and Hybridization of the Probes

There are several valid methods to prepare the DNA with the quality that is required to perform the MLPA (30–50 ng/μL, 260/280 Abs ratio of ~1.8). We routinely purify the DNA from 200μL of whole EDTA blood using the QIAamp DNA Blood Midi Kit and the Qiagen Biorobot QIAcube. A detailed protocol “Purification of DNA from Whole Blood Using the QIAamp DNA Blood Midi Kit” is provided at the Qiagen website (http://www. qiagen.com) (see Notes 2 and 3). The MLPA assay protocol described here refers to the SALSA MLPA kit P236-A3 ARMD probemix and it is available at the MRC Holland website (http://www.mlpa.com). Briefly, 1. Thaw and vortex buffers and probemix before use. 2. Dilute 1.5μL of the DNA sample [50 ng/μL] with 3.5μL of 1 TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.2). 3. Place tube or plate in the thermocycler and denature the DNA samples at 98  C for 5 min. Then cool them to 25  C for 1 min before adding to each sample 3μL mix containing 1.5μL SALSA MLPA+Probemix and 1.5μL SALSA MLPA buffer. 4. Mix, incubate at 95  C, 1 min and then at 60  C 16–20 h.

Ligation

Prepare the “ligase mix” by adding, 3μL of Ligase buffer A and 3μL Ligase buffer B to a tube containing 25μL H2O. Mix gently and add 1μL SALSA Ligase 65 enzyme, mix again. 1. Bring the thermocycler from 60  C to 54  C and add 32μL of the “ligase mix” into each tube without removing the samples from the thermocycler. Mix gently, do not use vortex (see Note 4). 2. Incubate the samples at 54  C for 15 min. Heat-inactivate at 98  C for 5 min and allow to cool to 20  C. At this point the tubes can be removed from the thermocycler.

PCR Reaction

Prepare the necessary amount of the Polymerase Master Mix by adding 7.5μL H2O, 2μL SALSA PCR primer mix and 0.5μL SALSA polymerase for each reaction. Mix well before use. Do not vortex. 1. Place the tubes in the thermocycler at 60  C for 1 min. Add 10μL Polymerase Master Mix to each tube, mix gently by pipetting. Do not vortex (see Note 4). 2. Run the PCR reaction as follows: 35 cycles (95  C, 30 s, 60  C, 30 s, and 72  C, 20 min).

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3. Cool the mixture to room temperature (or 15  C). 4. After the PCR reaction, do not open tubes in the room of the thermocycler. To avoid contamination, use different micropipettes for performing MLPA reactions and handling MLPA PCR products. PCR product can be stored at 4  C for 1 week. For longer periods, store between 20  C. As fluorescent dyes are light-sensitive, store PCR products in a dark box or wrapped in aluminum foil. 3.1.3 Analysis by Capillary Electrophoresis

The PCR fragments can be analyzed by capillary electrophoresis in an ABI Prism (3730) instrument as follows: 1. Mix 2μL of each PCR reaction product with 0.2μL LIZ GeneScan 500 size Standard (or alternatively, 0.3μL of ROX GeneScan 500) and 9.8μL formamide. 2. Denature at 95  C for 5 min, cool at 4  C for 5 min, then bring to room temperature. 3. The samples are loaded in the ABI Prism (3730) instrument and are run with the module GenMapper50_POP7_1. The system generates an .fsa file with the data. 4. The GeneMapper (ABI) software is used for a preliminary quality control analysis of the electropherograms.

3.1.4 Analysis by Coffalyser Software

The Coffalyser software and manual are available at the MRC Holland website (http://www.mlpa.com).

Setting the Parameters for the Analysis

The information of the probes used in the section called “sheet library” must be configured. P236 probes, which are commercial, are selected from a list. In the case of “custom” probes, select P200 and add the requested information about the design of the probes. In the section called “CE device,” it is also necessary to configure the capillary electrophoresis (CE) system data that is used. This initial configuration (sheet library and CE device) is only filled once; the data will be stored for further analysis under the same conditions.

Analysis of .fsa Files Obtained After Capillary Electrophoresis

A project is created for each of the processed probemix. To generate a new experiment within the project, give a name and indicate the CE device used. Select the type of experiment (DNA/MLPA), the sheet (i.e., P236) and the size marker (GS500-250) used. The .fsa files corresponding to the previously selected sheet are loaded, assigning to the controls the reference category. The samples are analyzed individually. Samples that meet the minimum conditions necessary for comparison with the reference are analyzed (Fragment MLPA Reaction Score > 1).

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Interpretation of the Results (Ratio Chart)

In each sample, for each of the probes, the results are shown by a blue box that indicates the dispersion between the references. A circle indicates the value of the sample (Fig. 4). To merge the results of the analysis obtained from both, commercial (P236) and custom (P200) experiments; use the numerical data of the “sample report” tab available after the final comparative analysis. To do this, select the column called “final ratio,” copy/paste to an Excel table and make a graphical representation of these values. Several examples of this graphical representation merging data from commercial (P236) and custom (P200) experiments are shown in Fig. 5.

3.2 Identification of CNVs in the Data Generated from NGS Gene Panels

The detection of CNVs requires a high depth and uniformity of coverage. Specific NGS gene panels that focus on groups of genes or regions of interest allow for greater coverage for a fixed amount of sequence than the current exome or genome sequencing (see Note 5). Regarding the sequencing method, we recommend capture methods based on hybridization with probes instead of multiplex PCR amplification methods (see Note 6). As described above, the CFH/CFHR1-5 gene region includes a number of large segmental duplications including different exons of the CFH and CFHR1-5 genes. Our NGS gene panel was designed so that these regions have saturation of probes. This improves the capture performance and therefore the alignment in the regions of interest. For enrichment of the NGS libraries we have used the Illumina kit “Nextera Custom Enrichment capture” following the manufacturer’s specifications. The NGS was done on an Illumina equipment (see Note 7). All these resulted in an excellent depth and uniformity of coverage in the CFH/CFHR1-5 gene region, with a depth greater than 500 on average and a 100% coverage of the target region. Once the fastq files are obtained, prior to generate the input files for computational analysis of CNV (mean20 file), the sequences have to be filtered in order to eliminate low “quality/ reliability” reads. Finally, the CNV calling defines the probability of “Deletion,” “Duplication,” “Normal,” or “Uncalled” (due to low coverage) of the potential CNV situations. To obtain this probability, the commonly used computational tools compare the coverage of the sequencing data obtained for a sample case with that from a control (or group of controls) (see Note 8). Here we will describe the computational analysis of CNV using the NextGENe software from Softgenetics (https://softgenetics.com/NextGENe.php) and the ONCOCNV package [32]. We use both of them to get a paired CNV analysis. Other commonly used CNV tools are m-HMM [33] and MSeq-CNV [34].

3.2.1 NextGENe

The NextGENe software runs on a Windows® operating system, which provides a GUI (graphical user interface), and does not require scripts or other bioinformatics support.

Fig. 4 “Ratio chart” representation. Figure shows the MLPA analysis by Coffalyser software of one sample carrying a CFHR3-CFHR1 deletion in heterozygosis, using commercial probes (P236) (a) and custom probes (P200) (b). In red are shown the regions with heterozygous deletions (Final ratio ¼ 0.5)

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Fig. 5 Common RCA gene cluster rearrangements. Each panel correspond to the excel graphical representation of the merged “final ratio” columns from the experiments using commercial probes (P236) and custom probes (P200) of the same DNA sample. Duplicated probes and probes that include common polymorphisms were excluded. The top right and left panels are control samples. Below are the MLPA results of DNA samples carrying different RCA gene cluster rearrangements. To help the interpretation of the data, a diagram of the chromosomal structure of both chromosomes is provided for each sample with the genes depicted in different colors

The NextGENe tool calculates the CNV calls based on the changes in coverage between the sample being tested and the control (or group of controls). This calculation includes the measurement of background noise and it is based on a statistical model based on HMM (Hidden Markov Model). Each region finally obtains a score that describes the probability of the CNV states “Deletion,” “Duplication,” and “Normal” (Table 2). Deletion calls are more confident than duplication calls because of the expected heterozygous deletion ratio (0.33) is farther away from the normal ratio (0.5) than the heterozygous duplication ratio (0.6). Running the NextGENe CNV Tool

1. With the filtered fastq files, mean20 files, generate the files .pjt (project) for each sample. 2. Open NextGENe viewer. 3. On the Comparisons menu, select CNV Tool.

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Table 2 Expected ratio table from copy number of the sample against control (Adapted from Softgenetics) Type

Sample

Control

Total

Expected Ratio

Homozygous duplication

4

2

6

4/6 ¼ 0.66

Heterozygous duplication

3

2

5

3/5 ¼ 0.60

Normal

2

2

4

2/4 ¼ 0.50

Heterozygous deletion

1

2

3

1/3 ¼ 0.33

Homozygous deletion

0

2

2

0/2 ¼ 0

4. When the CNV Tool window opens, the Method Selection tab is the first active tab. Select Dispersion and HMM. Leave Normalized Counts (recommended) selected or select RPKM. 5. Open the Data Input tab and fill input cells with “Sample.pjt” and “control.pjt” (or group of controls, in which case, Best match, average, or median choice are available) (see Note 8). 6. Load .bed file into basic settings tab, to define region of interest. 7. In advanced Setting tab we use the default Dispersion HMM setting. (Automatic fitting was used with 5% expected CNV, 100 minimum reads, and 20 minimum region length). For more information about Dispersion HMM see NextGENe CNV Detection—Dispersion and HMM (https:// softgenetics.com/NextGENe_013.php). 8. Results are visualized as a table or a graphical representation. The table, which can be exported, includes information of all the regions included in the NGS panel. The CNV Report block button (at the top of the table) groups adjacent regions with the same CNV call in the same row. This just shows the chromosomal position and length of a “deletion,” “normal,” or “duplicate” fragment. The graphical representation allows a more intuitive visualization of the CNV results (Fig. 6c). In addition, by pressing the Highligt ROI button, you can individually select each chromosome included in the design of your NGS panel to see each of the coverage-relationship data. 3.2.2 ONCOCNV

The ONCOCNV [32] package runs on any LINUX distribution, a minimal knowledge of code programming is required to work with. ONCOCNV performs a statistical calculation from the aligned sequencing readings to determine the number of copies with respect to a control (or a pool of controls). Each region finally obtains a score that describes the probability of the CNV states “Deletion”, “Duplication” and “Normal” (Table 3).

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Fig. 6 Comparison of the three analyses presented in the CFH cluster. Results of: A MLPA; B ONCOCNV; C NextGENe. Analyzed DNA sample carries a homozygote deletion of CFHR3 and CFHR1 and a heterozygote deletion of CFHR5 exons 4 to 9

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Table 3 Expected ratio table from copy number of the sample against control

Running the ONCOCNV Tool

Type

Sample

Control

Result (Ratio)

Homozygous duplication

4

2

0.7

Heterozygous duplication

3

2

0.4

Normal

2

2

0

Heterozygous deletion

1

2

0.7

Homozygous deletion

0

2

3

1. With the filtered fastq files, mean20 files, generate the files . bam for each sample. 2. Open “ONCOCNV.sh” with a text editor (gedit, textpad, etc.). 3. Set correct paths and file names in the top part of the “ONCOCNV.sh” (see Note 8). 4. Check properties of “ONCOCNV.sh” chmod + rwx. 5. Check formats: (a) Reads should be given in. BAM format. (b) Region coordinates should be given in .bed format (with or without the headline) and have region ID in column 4 and gene symbol in column 6 (e.g., chr1 2488068 2488201 AMPL223847 0 TNFRSF14). It is mandatory to provide gene names in the sixth column. (VERY IMPORTANT) Ensure the following: l

There are no duplicates in the coordinates.

l

Coordinates are sorted.

l

Gene names are gene names in the sense that corresponding regions fall in the same genomic locus and not on different chromosomes.

l

Gene names cannot be the same as region names or IDs because ONCOCNV assumes to have several regions per gene.

6. Run “ONCOCNV.sh” from the command line. 7. There are four output files per sample, one image file .png and three text files .txt. (a) The image file is a visual representation of normalized and annotated copy number profile. (b) The .profile.txt text file contains the predictions per region.

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(c) The .summary.txt text file contains the predictions per gene. (d) The .output.txt text file contains a break points summary. 8. “cat ONCOCNV_src_directory/perChrVisualization.R | R -slave --args directory_out/Sample.profile.txt chr_selected” will create an image file which include all the genes studied in the selected chromosome (Fig. 6b). 3.3 MLPA Versus Identification of CNVs in the Data Generated from NGS Gene Panels 3.3.1 Pros and Cons

The results obtained with the three methods (MLPA, ONCOCNV, and NextGENe) for a sample carrying a homozygote deletion of CFHR3 and CFHR1 and a heterozygote deletion of CFHR5 exons 4 to 9 (Fig. 6) are shown for comparison. 1. MLPA and CNV from NGS are complementary techniques. 2. Gene conversion events can only be resolved by MLPA. 3. CNV analysis provides precise information of breaking points. 4. The hybridization of the probes in the MLPA may be affected by the presence of SNPs in that region, affecting the interpretation of the results. In contrast, analysis of CNVs from data generated by NGS with capture-enriched libraries is not affected by the presence of SNPs. 5. The difficulties in the alignment of short reads in very high homology regions in the RCA cluster is a problem when using CNV analysis from NGS data.

4

Notes 1. To complement the regions covered by the MLPA SALSA MLPA kit P236-A3 ARMD we recommend designing an additional set of probes targeting regions that are not included the ARMD probemix (i.e., CFHR4). This additional set of probes, mixed together with the control probes (P200) provided by MRC-Holland, will be used in an independent MLPA experiment. 2. The purity, concentration and integrity of the genomic DNA samples are very important. We recommend that all the samples analyzed in the same assay are purified with the same method. If the DNAs are obtained by different methodologies we recommend to repurify them with the Kit NucleoSpin gDNA Cleanup (Macherey-Nagel). Check DNA integrity in an agarose gel. Purification methods based on magnetics beads are not recommended. 3. Reference samples must be included in each MLPA experiment. They are needed to normalize the signal of the probes

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within a given MLPA run. The software analysis needs at least three different reference samples per MLPA run. 4. Working with more than 16 samples is not recommended, as it may cause time delays and artefacts. 5. In order to have a reliable CNV result a high coverage is recommended. We recommend coverages higher than 500. 6. For enrichment, hybridization capture based methods have demonstrated better coverage uniformity in comparison to amplicon enrichment–based methods [35, 36]. 7. It is recommended to make large size libraries, around 500 nt, and run the samples in Illumina Miseq with V2 (250  2) or V3 (300  2) chip, because the longer the reads, the better the assembly. These longer reads are particularly important to resolve regions of high homology. 8. The controls used in the comparison must be of the same sequencing run, otherwise a high dispersion could be observed in the comparison, diminishing the sensibility and increasing the possibility of false negatives.

Acknowledgments SRdeC is supported by the Spanish “Ministerio de Economı´a y Competitividad-FEDER” (SAF2015-66287R, PID2019104912RB-I00, RTC-2016-4635-1 and the Autonomous Region of Madrid (S2017/BMD-3673). Secugen has received a soft loan from the Spanish “Ministerio de Economı´a y Competitividad” Retos Program RTC-2016-4635-1 cofinanced by FEDER funds. References 1. Rodrı´guez De Co´rdoba S, Dı´az-Guille´n MA, ˜ er D (1999) An integrated map of Heine-Sun the human regulator of complement activation (RCA) gene cluster on 1q32. Mol Immunol 36:803–808. https://doi.org/10.1016/ S0161-5890(99)00100-5 2. Lambert J-C, Heath S, Even G et al (2009) Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer’s disease. Nat Genet 41:1094–1099. https://doi.org/10.1038/ng.439 3. Venables JP, Strain L, Routledge D et al (2006) Atypical haemolytic uraemic syndrome associated with a hybrid complement gene. PLoS Med 3:e431. https://doi.org/10.1371/jour nal.pmed.0030431 4. Valoti E, Alberti M, Tortajada A et al (2014) A novel atypical hemolytic uremic syndrome-

associated hybrid CFHR1/CFH gene encoding a fusion protein that antagonizes factor H-dependent complement regulation. J Am Soc Nephrol 26:209–219. https://doi.org/ 10.1681/ASN.2013121339 5. Francis NJ, McNicholas B, Awan A et al (2012) A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome. Blood 119:591–601. https://doi.org/10. 1182/blood-2011-03-339903 6. Goicoechea de Jorge E, Tortajada A, Pinto Garcı´a S et al (2018) Factor H competitor generated by gene conversion events associates with atypical hemolytic uremic syndrome. J Am Soc Nephrol 29:1–10. https://doi.org/10. 1681/ASN.2017050518

Detection of Genetic Rearrangements in the RCA Cluster 7. Gharavi AG, Kiryluk K, Choi M et al (2012) Genome-wide association study identifies susceptibility loci for IgA nephropathy. Nat Genet 43:321–327. https://doi.org/10.1038/ng. 787.Genome-wide 8. Hughes AE, Orr N, Esfandiary H et al (2006) A common CFH haplotype, with deletion of CFHR1 and CFHR3, is associated with lower risk of age-related macular degeneration. Nat Genet 38:1173–1177. https://doi.org/10. 1038/ng1890 9. Tortajada A, Ye´benes H, Abarrategui-Garrido C et al (2013) C3 glomerulopathy–associated CFHR1 mutation alters FHR oligomerization and complement regulation. J Clin Invest 123:2434–2446. https://doi.org/10.1172/ JCI68280DS1 10. Krych-Goldberg M, Atkinson JP (2001) Structure-function relationships of complement receptor type 1. Immunol Rev 180:112–122 11. Liu D, Niu ZX (2009) The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35). Immunopharmacol Immunotoxicol 31:524–535. https://doi.org/10. 3109/08923970902845768 12. Klickstein LB, Wong WW, Smith JA et al (1987) HUMAN C3b/C4b RECEPTOR (CR1) demonstration of long homologous repeating domains that are composed of the short consensus repeats characteristic of C3/C4 binding proteins. J Exp Med 165:1095–1112. https://doi.org/10.1084/ jem.165.4.1095 13. Rodriguez de Cordoba S, Rubinstein P (1986) Quantitative variations of the C3b/C4b receptor (CR1) in human erythrocytes are controlled by genes within the regulator of complement activation (RCA) gene cluster. J Exp Med 164:1274–1283. https://doi.org/ 10.1084/jem.164.4.1274 14. Brouwers N, Van Cauwenberghe C, Engelborghs S et al (2012) Alzheimer risk associated with a copy number variation in the complement receptor 1 increasing C3b/C4b binding sites. Mol Psychiatry 17:223–233. https://doi. org/10.1038/mp.2011.24 15. Kisserli A, Tabary T, Cohen JHM et al (2017) High-resolution melting PCR for complement receptor 1 length polymorphism genotyping: an innovative tool for Alzheimer’s disease gene susceptibility assessment. J Vis Exp 125:1–11. https://doi.org/10.3791/56012 16. Rodriguez de Cordoba S, Hidalgo MS, Pinto S, Tortajada A (2014) Genetics of atypical hemolytic uremic syndrome (aHUS). Semin

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Thromb Hemost 40:422–430. https://doi. org/10.1055/s-0034-1375296 17. Jo´zsi M, Tortajada A, Uzonyi B et al (2015) Factor H-related proteins determine complement-activating surfaces. Trends Immunol 36:374–384. https://doi.org/10. 1016/j.it.2015.04.008 18. Pe´rez-Caballero D, Gonza´lez-Rubio C, Gallardo ME et al (2001) Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome. Am J Hum Genet 68:478–484. https://doi.org/10. 1086/318201 19. Tortajada A, Gutie´rrez E, Goicoechea de Jorge E et al (2017) Elevated factor H–related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy. Kidney Int 92:953–963. https://doi.org/10.1016/j.kint.2017.03.041 20. Zhao J, Wu H, Khosravi M et al (2011) Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility. PLoS Genet 7:1–9. https://doi.org/10.1371/jour nal.pgen.1002079 21. Abarrategui-Garrido C, Martı´nez-Barricarte R, Lo´pez-Trascasa M et al (2009) Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome. Blood 114:4261–4271. https://doi.org/10.1182/blood-2009-05223834 22. Jodele S, Licht C, Goebel J et al (2013) Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy. Blood 122:2003–2007. https://doi.org/10.1182/blood-2013-05501445 23. Moore I, Strain L, Pappworth I et al (2010) Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome. Blood 115:379–387. https://doi.org/ 10.1182/blood-2009-05-221549 24. Zipfel PF, Edey M, Heinen S et al (2007) Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome. PLoS Genet 3:0387–0392. https://doi.org/10. 1371/journal.pgen.0030041 25. Eyler SJ, Meyer NC, Zhang Y et al (2013) A novel hybrid CFHR1/CFH gene causes atypical hemolytic uremic syndrome. Pediatr Nephrol 28(11):2221–2225. https://doi. org/10.1007/s00467-013-2560-2

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26. Chen Q, Wiesener M, Eberhardt HU et al (2014) Complement factor H-related hybrid protein deregulates complement in dense deposit disease. J Clin Invest 124:145–155. https://doi.org/10.1172/JCI71866 27. Malik TH, Lavin PJ, Goicoechea de Jorge E et al (2012) A hybrid CFHR3-1 gene causes familial C3 glomerulopathy. J Am Soc Nephrol 23:1155–1160. https://doi.org/10.1681/ ASN.2012020166 28. Xiao X, Ghossein C, Tortajada A et al (2016) Familial C3 glomerulonephritis caused by a novel CFHR5-CFHR2 fusion gene. Mol Immunol 77:89–96. https://doi.org/10. 1016/j.molimm.2016.07.007 29. Gale DP, Goicoechea de Jorge E, Cook HT et al (2010) Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis. Lancet 376:794–801. https://doi.org/ 10.1016/S0140-6736(10)60670-8 30. Medjeral-Thomas N, Malik TH, Patel MP et al (2014) A novel CFHR5 fusion protein causes C3 glomerulopathy in a family without Cypriot ancestry. Kidney Int 85:933–937. https://doi. org/10.1038/ki.2013.348 31. Togarsimalemath SK, Sethi SK, Duggal R et al (2017) A novel CFHR1-CFHR5 hybrid leads to a familial dominant C3 glomerulopathy.

Kidney Int 92:876–887. https://doi.org/10. 1016/j.kint.2017.04.025 32. Boeva V, Popova T, Lienard M et al (2014) Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data. Bioinformatics 30:3443–3450. https://doi.org/10.1093/bio informatics/btu436 33. Wang H, Nettleton D, Ying K (2014) Copy number variation detection using next generation sequencing read counts. BMC Bioinformatics 15:109. https://doi.org/10.1186/ 1471-2105-15-109 34. Malekpour SA, Pezeshk H, Sadeghi M (2018) MSeq-CNV: accurate detection of copy number variation from sequencing of multiple samples. Sci Rep 8:1–12. https://doi.org/10. 1038/s41598-018-22323-8 35. Samorodnitsky E, Datta J, Jewell BM et al (2015) Comparison of custom capture for targeted next-generation DNA sequencing. J Mol Diagn 17:64–75. https://doi.org/10.1016/j. jmoldx.2014.09.009 36. Samorodnitsky E, Jewell BM, Hagopian R et al (2015) Evaluation of hybridization capture versus amplicon-based methods for wholeexome sequencing. Hum Mutat 36:903–914. https://doi.org/10.1002/humu.22825

Chapter 17 Complement Detection in Mouse Kidneys by Immunofluorescence Jennifer Laskowski and Joshua M. Thurman Abstract Immunofluorescence staining of tissues has become a reliable and informative technique used in a diverse set of applications, ranging from simple detection of an antigen of interest in a specific location to the semiquantitative analysis of spatial relationships between multiple antigens and/or cell types. During complement activation, circulating complement proteins are covalently fixed to target tissues, providing a durable marker of complement activation in the tissue, and many of these proteins can be readily detected by immunofluorescence microscopy. In general, staining for complement fragments is much like staining for other noncomplement epitopes. However, one key difference is the diligence with which unfixed tissues must be handled when staining for complement fragment. Here we explain the process of dual staining frozen mouse kidney sections for the complement proteins C3 and C4. Throughout the protocol, we will emphasize important steps for preserving complement protein integrity as well as tips to improve the signalto-noise ratio to improve overall image quality. Key words Complement, Immunofluorescence, Formalin-fixed and paraffin-embedded, Microscopy, Autofluorescence

1

Introduction The complement cascade is comprised of more than 30 circulating and cell surface proteins. One of the unique characteristics of the complement system is that during activation circulating proteins become covalently fixed to nearby cell and tissue surfaces. This generates a durable “footprint” of activation within the tissue. Immunofluorescence microscopy allows for the simple detection of an antigen of interest in a specific location to the semiquantitative analysis of spatial relationships between multiple antigens and/or cell types [1–3]. This method can be used to detect complement proteins in tissue biopsies from human patients and in tissue samples from in vitro systems and animal models. The detection of complement proteins by immunofluorescence microscopy is now routinely performed on clinical biopsies. Indeed, the detection

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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of complement components within tissues by immunofluorescence is central to the diagnosis of many diseases, including lupus nephritis and C3 glomerulopathy [4, 5]. Detection of complement proteins by immunofluorescence microscopy provides insight into several aspects of the underlying disease process. Detection of C3, the central component of the complement cascade, is a good method of detecting complement activation by any of the activation pathways (classical pathway, mannose binding lectin pathway, or alternative pathway). By microscopy one can identify where in the tissue the activation occurred, often identifying the specific ultrastructural location of activation. By staining for different complement proteins, such as C1q, C4, or the mannose binding lectins, one can also determine which activation pathways are involved [6]. Detection of C4d in transplant biopsies has become an important tool for diagnosing antibody-mediated rejection [7], and in native kidney biopsies it is a marker of immune-complex mediated disease [8]. The development of new antibodies specific for various complement activation fragments will further expand the information that immunofluorescence microscopy can reveal about disease pathogenesis. Dual staining for complement proteins in combination with other proteins can further aid in localizing the site of complement activation. In the current protocol we describe a method for costaining tissue for complement C3 and C4 (Fig. 1). Depending on the goal of the experiment, antibodies to other complement proteins or tissue markers can be used.

2

Materials All reagents should be prepared with ultrapure water and stored at 4
C, unless otherwise indicated.

2.1 Frozen and FFPE Tissues

1. Hydrophobic pen. 2. Timer. 3. 1 PBS chilled to 4
C. 4. Blocking buffer: 1 PBS, 5% heat-inactivated normal goat serum (NGS) or heat-inactivated fetal bovine serum (FBS) (see Note 1), 1% bovine serum albumin (BSA) (fraction V, heat-shock treated). Sterile-filter with 0.22 μm filter and store at 20
C. 5. Antibody diluent buffer: 1 PBS, 2% heat-inactivated NGS or FBS, 1% BSA (fraction V, heat-shock treated). Sterile-filter with 0.22 μm filter and store at 20
C. 6. Humidified chamber: empty pipette tip box with platform (see Note 2).

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Fig. 1 Complement proteins C3 and C4 in C57Bl/6 kidney. Frozen kidney (5 μm) sections from a 9-month-old C57Bl/6 male mouse were stained according to protocol and imaged with an Olympus FV-1000 confocal microscope. Top panels shown at 20 magnification; bottom panels shown at 60 magnification. C4 can be seen in the glomeruli (arrowheads), indicating activation of the classical or lectin pathway. C3 in the absence of C4 can be seen at other locations in the tubulointerstitium (small arrows), suggesting activation of the alternative pathway at this location

7. Fitc goat IgG to mouse complement C3. 8. Rat anti-mouse C4, clone 16D2. 9. Alexa Fluor 647 goat anti-rat IgG (heavy and light chains). 10. DAPI (40 ,6-diamidino-2-phenylindole) diluted to 1 μg/ml in 1 PBS. 11. Microscope cover glass—#1.5 (0.16–0.19 mm thickness, refractive index ¼ 1.515) borosilicate glass (see Note 3). 12. Mounting medium: equal parts 1 PBS and glycerol (molecular biology grade), stored at room temperature. 13. Slide sealant: nonfluorescing clear fingernail polish. 14. Orbital rocker: optional. 2.2 Frozen Tissues Only

1. Slides with frozen kidney section(s), 4–6 μm thick: stored at 80
C. 2. Ice-cold absolute acetone: certified ACS quality, stored at 20
C.

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3. Sudan Black B (SBB): 0.05% diluted in 70% EtOH (see Notes 4–6). 4. Cotton swabs. 2.3 FFPE Tissues Only

1. Slides with formalin-fixed, paraffin-embedded kidney sections, 3–5 μm thick. 2. Xylenes (reagent grade): 2–100 ml individual aliquots (labeled “xylene 1” and “xylene 2”) (see Note 7). 3. Ethanol (200 proof, anhydrous) for rehydration gradient: 100 ml each of 100%, 95%, 90%, and 70% EtOH. 4. Glass Coplin staining jar(s) with lid: 65 ml capacity or greater. 5. Antigen Retrieval Buffer: 10 mM citric acid (anhydrous, 192.12 g/mol), 0.05% Tween 20, pH 6.0. For 1 L, add 1.92 g citric acid and 500 μl Tween 20 to 950 ml ultrapure water allowing for complete suspension of both reagents. Adjust pH to 6.0 with 5 N HCl and add remaining volume of ultrapure water to reach a final volume of 1 L. Pass buffer through a 0.45 μm PES filter and store at 4
C for up to 12 months. Discard if turbidity develops. 6. Water bath capable of sustaining 95
C for 30 min. 7. Slide mailers (used for antigen retrieval): We use the LockMailer slide containers from Market Lab.

3

Methods

3.1 IF Using Frozen Tissue Sections

1. Remove slides from 80
C storage and place on paper towels or some other flat, absorbent surface. Allow slides to warm to room temperature and for all condensation to resolve (see Note 8). 2. Add 0.5–1 ml ice cold acetone to each slide, ensuring complete coverage of all tissue. Allow tissues to “fix” for 5 min, pipetting additional acetone onto the tissue sections as it evaporates from surface and tissues begin to dry. At the end of 5-min fixation, allow acetone to evaporate completely and tissues to dry. 3. Apply 200–250 μl cold PBS to each kidney section, making sure all tissue is covered. Incubate for 2 min and remove by aspirating with pipettor or vacuum-aspiration device. 4. Repeat step 3. When removing PBS following second incubation, make sure the slide surface immediately surrounding the tissue (approximately 1 cm) is completely dry (see Note 9). 5. Draw a hydrophobic boundary around tissue section, approximately 0.5–1 cm out from tissue border. Carefully apply a small volume of cold PBS to the tissue, making sure the entire section is covered. Do not allow PBS to encroach the newly drawn boundary until it has dried completely (see Note 10).

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Fig. 2 A humidified chamber can be easily and efficiently made by adding 50 ml of deionized or ultrapure water to the base of a used, clean tip box. When the box lid is closed, evaporation is nearly eliminated

6. Prepare humidified chamber: remove the empty tip platform, add 50 ml DI water to the bottom of the tip box, and replace platform (Fig. 2). Gently place slides on the tip platform. 7. After 10–15 min, check hydrophobic boundary by lightly touching the outer edge. 8. Aspirate PBS from each tissue section. Individually tilt each slide 30–45
to pool remaining PBS against hydrophobic boundary and aspirate remaining PBS (Fig. 3). 9. Apply blocking buffer in a volume sufficient to completely cover the tissue (approximately 200 μl). Carefully place the lid on the tip box and allow slides to incubate at room temperature for a minimum of 1 h. If blocking for longer than 2 h, do so at 4
C. 10. During this incubation, prepare primary antibodies by diluting Fitc goat IgG to mouse C3 (10 μg/ml) and anti-mouse C4 (2 μg/ml) together in antibody diluent buffer. Protect from light by wrapping in aluminum foil and storing at 4
C until ready to apply to tissues. 11. Remove blocking buffer by pipetting or aspiration. Apply cold PBS to each section, making sure to completely cover tissue. Incubate for 2–3 min. 12. Repeat step 9. Tilt each slide 45
and aspirate remaining PBS from slide surface.

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Fig. 3 Tilting slides at a 30–45
angle forces excess buffer to pool (yellow arrows) at the bottom of the hydrophobic boundary (highlighted with yellow dashes). Complete removal of buffer will prevent unintentional dilution of antibodies during staining. Removal of 0.05% SBB/70% EtOH with cotton applicator is shown above

13. Apply primary antibody, once again making sure entire tissue surface is covered. Because this antibody is directly conjugated, protect slides from light by covering tip box with aluminum foil. Incubate on bench at room temperature for a minimum of 1 h, or overnight at 4
C. 14. Aspirate primary antibody from tissue sections as in step 11. Repeat 2 additional times, making sure to remove all PBS after third wash by tilting slides 45
. 15. Apply secondary antibody mixture to each section, protect from light and incubate for 1 h at room temperature. 16. Repeat step 13. 17. * Optional step: Apply diluted DAPI to each tissue section, protect from light and incubate for 5 min at room temperature (see Note 11). 18. Repeat step 13. Make sure all PBS is removed from tissue and the area within the hydrophobic boundary. 19. To reduce endogenous autofluorescence in the tissue, carefully apply the 0.05% Sudan Black B in 70% EtOH [9] directly to the tissue section (see Note 12, Fig. 4). Incubate for 15 min, protected from light at room temperature. 20. During this incubation, fill a Coplin jar with enough ultrapure water to completely submerge the stained tissues on each slide. 21. Carefully remove Sudan Black B by slightly (25–30
) tilting the slide and absorbing the solution that collects in between the tissue and the hydrophobic boundary with a cotton swab,

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Fig. 4 Sudan Black B (SBB) minimizes endogenous autofluorescence in frozen kidney tissue. A comparison of autofluorescence in C57Bl/6 serial kidney sections between Sudan Black B-treated and untreated tissue is shown above. Sudan Black B (0.05%) was applied to tissue in the first panel (+SBB), significantly reducing the amount of observable background staining. Autofluorescence is readily observed in the tubules of the untreated tissue (SBB). Images acquired with an Olympus FV1000 confocal microscope and are shown at 10 magnification

taking care to avoid touching the tissue (Fig. 3). Place the slide in Coplin jar with water. Repeat for remaining slides. 22. Wash slides for 10 min, with periodic gentle agitation of the Coplin jar. 23. Discard the water and replace with fresh ultrapure water. Repeat step 21. 24. To mount and seal slides, take 1 slide from Coplin jar and remove as much excess water as possible from the surfaces outside of the hydrophobic boundary (including the back of the slide) with paper towel or Kim wipes and aspirator. Without drying tissue, remove excess water from the area immediately surrounding the tissue. Place slide on a flat surface on top of a clean paper towel. Apply 5–10 μl mounting medium to the cover glass, invert and gently drop cover glass at a 45
angle onto tissue section, so that the mounting medium spreads outward from the center of the tissue. Remove any air bubbles from underneath the cover glass (see Note 13) and seal all edges of the slide with a thin layer of clear fingernail polish (see Note 14). 25. Place sealed slides in a slide box, resting on top of slide dividers (Fig. 5) and allow nail polish to completely dry. Wipe the bottom surface of each sealed slide with a Kim wipe or soft paper towel that has been lightly sprayed with 70% EtOH (this will remove any buffers or mounting medium that may have been transferred to the slide during the staining, washing or mounting processes). Protect slides from light by placing in

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Fig. 5 Freshly sealed slides are dried in a slide box, resting on the tops of the slide dividers, minimizing contact between the slide edges and box

slide box (traditional orientation) and storing at 4
C until ready to image (see Note 15). 3.2 IF Using Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue Sections

l

Xylene is used in the deparaffinization and rehydration process. Due to the health and safety dangers associated with xylene, it is important become familiar with proper handling and disposal, as well as the proper protective equipment that should be worn when handling xylene (see Note 16).

l

Deparaffinization and rehydration overview (5 min each): Xylene #1—Xylene #2–100% EtOH—95% EtOH—90% EtOH—70% EtOH—H2O—H2O. 1. Start water bath warming to 95
C. Place a 100–200 ml beaker with 50 ml of deionized water in the water bath. Fill slide mailer with antigen retrieval buffer (approximately ¾ full) and place in the beaker in the water bath. 2. Set up for deparaffinization and rehydration of tissues in a clean, vacant chemical fume hood. Place at least two Coplin jars, 2–100 ml xylene aliquots, EtOH gradient (100–70%) aliquots, several clean paper towels, and a timer inside chemical fume hood. Preset timer for 5 min. Place a third Coplin jar, filled with DI water, near a laboratory sink. 3. Place slide(s) in empty Coplin jar and add just enough of “xylene 1” to submerge all tissue sections and leave for 5 min. Slides should be agitated twice each minute by gently shaking Coplin jar for several seconds. At the end of the 5 min, pour or pipette xylene into “xylene 1” bottle. Close and set aside (see Note 17). 4. Pour contents of “xylene 2” into Coplin jar. Repeat step 3.

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5. Transfer slides to clean Coplin jar and fill 100% EtOH, just enough to cover tissue sections. Repeat step 3. 6. Repeat step 3 with 95% EtOH. 7. Repeat step 3 with 90% EtOH. 8. Repeat step 3 with 75% EtOH. Once EtOH has been transferred back to the correct storage container, take slides (still in Coplin jar) to sink and place in the Coplin jar with DI water. Leave for 5 min, with periodic agitation. 9. Dispose of DI water and replace with fresh DI water. Leave for 5 min. 10. Carefully transfer slides to the prewarmed slide mailer containing antigen retrieval buffer. The water bath will be extremely hot. Extreme caution should be exercised during this step. Leave slides in hot water bath for 20 min. 11. Remove slide mailer from the water bath and allow it to cool on lab bench for 10 min. 12. Once cooled, remove slides from slide mailer and wash in running DI water for 3 min (see Note 18). 13. Dry slide(s), particularly the area immediately surrounding the tissue. 14. Follow steps 5–18, 24, and 25 in Subheading 3.1 (see Note 19).

4

Notes 1. Heat-inactivation of NGS or FBS used in blocking and antibody diluent buffers is good practice for any IF staining protocol, but a necessity when staining for complement fragments. To heat-inactivate serum, prepare a 56
C water bath with enough volume to completely submerge the serum. Place thawed serum in water bath for 30 min, gently agitating every 2–4 min to ensure uniform heat distribution throughout serum. Once complete, aliquot and store at 20
C. 2. We use empty pipette tip boxes with approximately 50 ml of DI water added to the bottom of the box as a humidified chamber. 3. The type and thickness of cover glass used is highly dependent on the specific microscopy modality being performed. In general, #1.5 cover glass will work with objectives used in most general microscopy applications. Specialized microscopy techniques may vary. 4. We suggest making a 0.1% stock solution by adding 0.05 g Sudan Black B to 50 ml 70% EtOH. Filter through 0.22 μm PES filter and store in a glass container, protected from light in

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fireproof flammables cabinet. To prepare 0.05% working buffer, combine equal volumes of 0.1% SBB stock solution with 70% EtOH. 5. If a stock solution greater than 0.1% is preferred, we suggest increasing the total volume to at least 250 ml. Stock solutions greater than 0.5% are not recommended. 6. Filtration of SBB solution is essential. Failure to do so may result in SBB aggregates collecting on the tissue surface, potentially obscuring fluorescence and diminishing image quality. 7. Buffers used for deparaffinization and rehydration can be reused for multiple staining sessions. Buffers should be stored individually in glass bottles and kept in a fireproof flammables cabinet. 8. Tissue must be fixed as quickly as possible. Allowing tissue to remain at room temperature prior to fixation could result in erroneous staining. 9. The area where the hydrophobic boundary will be drawn must be completely dry. If the slide is still wet, the boundary will not thoroughly adhere to the slide surface. 10. Depending on the thickness of the hydrophobic layer, it may take as long as 1 h to dry enough to continue with the protocol. It is critical that the tissue remain hydrated during this time and the area around the hydrophobic boundary to remain dry. 11. We suggest applying DAPI nuclear stain to kidney sections if the presence of the target antigen is unknown, the quantity of target antigen is very low, or if the spatial relationship between the target antigen and nuclei is required. Often, we find that in dense regions of nuclei (such as in the glomerulus), the DAPI stain obscures signal from the target antigen, particularly when in low quantity. 12. Take care to avoid applying Sudan Black B directly to the hydrophobic boundary. The high EtOH content may cause regions of the boundary to lift, making it difficult to block autofluorescence. When applied to center of the tissue, the Sudan Black B solution will spread outward and touch the hydrophobic boundary but will not breach it. 13. Using a clean 300 μl pipette tip, touch the tip to the cover glass to one side of the bubble. Gently apply pressure, forcing the bubble to the edge of the cover glass. 14. Several manufacturers produce a variety of 2-in-1 mounting/ sealant media, which eliminates the need to seal the edges of the slide. 15. Image stained slides as soon as possible or within 1–2 h. It is possible to leave slides at 4
C for longer periods of time;

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however, we find there is a risk of fluor decay when slides are left for several hours, particularly when using FITC. 16. Xylene is an extremely toxic aromatic hydrocarbon. Deparaffinization and rehydration series should be performed in a chemical fume hood and only while using the appropriate personal protective equipment (PPE) [10, 11]. 17. Quickly wipe up any spilled xylene and properly dispose of waste. 18. When washing slides in running DI water, tilt slides downward and allow water to make contact with the top of slide and run over tissue. This will prevent unintentional damage to the tissue. 19. Sudan Black B does not effectively reduce autofluorescence in FFPE tissues. References 1. Feng Z, Puri S, Moudgil T, Wood W, Hoyt CC, Wang C, Urba WJ, Curti BD, Bifulco CB, Fox BA (2015) Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma. J Immunother Cancer 3:47. https:// doi.org/10.1186/s40425-015-0091-z 2. Feng Z, Jensen SM, Messenheimer DJ, Farhad M, Neuberger M, Bifulco CB, Fox BA (2016) Multispectral imaging of T and B cells in murine spleen and tumor. J Immunol 196 (9):3943–3950. https://doi.org/10.4049/ jimmunol.1502635 3. Kefaloyianni E, Muthu ML, Kaeppler J, Sun X, Sabbisetti V, Chalaris A, Rose-John S, Wong E, Sagi I, Waikar SS, Rennke H, Humphreys BD, Bonventre JV, Herrlich A (2016) ADAM17 substrate release in proximal tubule drives kidney fibrosis. JCI Insight 1(13). https://doi. org/10.1172/jci.insight.87023 4. Fakhouri F, Fremeaux-Bacchi V, Noel LH, Cook HT, Pickering MC (2010) C3 glomerulopathy: a new classification. Nat Rev Nephrol 6(8):494–499. https://doi.org/10.1038/ nrneph.2010.85 5. Nossent H, Berden J, Swaak T (2000) Renal immunofluorescence and the prediction of renal outcome in patients with proliferative lupus nephritis. Lupus 9(7):504–510. https://doi.org/10.1177/ 096120330000900705 6. Renner B, Strassheim D, Amura CR, Kulik L, Ljubanovic D, Glogowska MJ, Takahashi K,

Carroll MC, Holers VM, Thurman JM (2010) B cell subsets contribute to renal injury and renal protection after ischemia/reperfusion. J Immunol 185(7):4393–4400. https:// doi.org/10.4049/jimmunol.0903239 7. Bohmig GA, Exner M, Habicht A, Schillinger M, Lang U, Kletzmayr J, Saemann MD, Horl WH, Watschinger B, Regele H (2002) Capillary C4d deposition in kidney allografts: a specific marker of alloantibodydependent graft injury. J Am Soc Nephrol 13 (4):1091–1099 8. Sethi S, Nasr SH, De Vriese AS, Fervenza FC (2015) C4d as a diagnostic tool in proliferative GN. J Am Soc Nephrol 26(11):2852–2859. https://doi.org/10.1681/ASN.2014040406 9. Sun Y, Yu H, Zheng D, Cao Q, Wang Y, Harris D, Wang Y (2011) Sudan black B reduces autofluorescence in murine renal tissue. Arch Pathol Lab Med 135 (10):1335–1342. https://doi.org/10.5858/ arpa.2010-0549-OA 10. Kandyala R, Raghavendra SP, Rajasekharan ST (2010) Xylene: an overview of its health hazards and preventive measures. J Oral Maxillofac Pathol 14(1):1–5. https://doi.org/10. 4103/0973-029X.64299 11. Rajan ST, Malathi N (2014) Health hazards of xylene: a literature review. J Clin Diagn Res 8 (2):271–274. https://doi.org/10.7860/ JCDR/2014/7544.4079

Chapter 18 Complement Detection in Human Tumors by Immunohistochemistry and Immunofluorescence Marie V. Daugan, Margot Revel, Laetitia Lacroix, Catherine Saute`s-Fridman, Wolf H. Fridman, and Lubka T. Roumenina Abstract Tumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of the cells. There is a high heterogeneity of the staining within tumors but also between patients. Semi-quantification of the staining in large cohorts of patients allows to investigate the prognostic impact of the local complement production and activation. Here we explain the staining process for C1q, C4, and C3 in human paraffin-embedded tumor sections by immunofluorescence and immunohistochemistry. Key words Complement, Immunohistochemistry (IHC), Immunofluorescence (IF), Tumors, Production, Activation, Prognostic

1

Introduction Tumors contain a complement rich environment [1] including a series of complement proteins, regulators, and receptors [2]. Numerous studies show that complement plays a key role in cancer progression in animal models and patients cohorts [3]. Negative prognostic impact of the positive staining for C1q and/or C4d were detected by our group in clear cell renal cell carcinoma (ccRCC) and in non–small cell lung cancer [4, 5]. Immunohistochemistry (IHC) is a robust method to study the localization (adjacent healthy tissue, tumor core, invasive margin, necrotic areas) and the density of the complement proteins within tumors. For C1q, C4, and C3, two types of staining are observed in Formalin-Fixed Paraffin-Embedded (FFPE) sections: a cytoplasmic one showing the local production and a membranous one reflecting

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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complement activation [4]. A high heterogeneity of complement staining is observed between tumors of patients but also within the same tumor, some areas being strongly positive others completely negative. This difference can be used to perform semiquantification and classify the patients into groups according to the density of positive cells. Ensuing survival analyses allow to evaluate the prognostic value of these proteins and may be useful to identify new biomarkers and targets in cancer. Within the TME, complement proteins can be produced by various cell types including immune cells, endothelial cells, fibroblasts, and tumor cells. The identification of complement producing cells can be performed by multiplex-immunofluorescence which provides sensible resolution, spatial information, and information about staining intensity. The complement cell map that is generated is very informative to identify the keys cell actors within the complement tumor microenvironment.

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Materials All reagents must be handled with care. Wear protective gloves/ protective clothing. Refer to safety datasheet to have more details on specific hazards.

2.1

Buffers

1. Ethanol solution: Ethanol absolute (VWR, ref. 20821). Dilute ethanol absolute in distilled water to make 90% (preparation for a final volume of 300 mL: in a 500 mL graduated cylinder add 270 mL of ethanol absolute and 30 mL of distilled water), 70% (preparation for a final volume of 300 mL: in a 500 mL graduated cylinder add 210 mL of ethanol absolute and 90 mL of distilled water) and 50% (preparation for a final volume of 300 mL: in a 500 mL graduated cylinder add 150 mL of ethanol absolute and 150 mL of distilled water) final solutions. 2. Clearene: Leica, ref-3803600 (ready to use). 3. Target retrieval solution: Dako Target Retrieval Solution pH 6, Citrate buffer (Dako, S236984-2) or pH 9, Tris/EDTA buffer (Dako, K800421-2). With a 2 L graduated cylinder add 1470 mL of distilled water and 30 mL of the retrieval solution (1 bottle). 4. Blocking buffer: Dako protein block, serum free (Dako, X0909, ready to use). 0.25% casein in PBS, containing stabilizing protein and 0.015 mol/L sodium azide (NaN3). 5. Antibody diluent: Antibody diluent, Dako real (Dako, S2022, ready to use). Tris buffer, pH 7.2, containing 15 mmol/L NaN3, and protein. 6. TBS buffer: 140 mM sodium chloride, 25 mM Tris, 3 mM potassium chloride, pH 7.4 in water. In a 1 L graduated cylinder add 100 mL of distilled water. Weigh 8.18 g of sodium

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chloride, 3.03 g of Tris, and 0.224 g of potassium chloride, transfer to the cylinder. Add distilled water to the volume of 900 mL. Mix well until all powders are dissolved, and adjust the pH with HCl (see Note 1). Make up to 1 L with distilled water and store at room temperature. 7. TBS buffer Tween 0.04%: 140 mM sodium chloride, 25 mM Tris, 3 mM potassium chloride, and Tween 20 0.04%, pH 7.4 in water. In a 2 L graduated cylinder add 100 mL of distilled water. Weigh 16.36 g of sodium chloride, 6.05 g of Tris, and 0.447 g of potassium chloride, transfer to the cylinder. Add distilled water to the volume of 1900 mL. Mix well until all powders are dissolved, and adjust the pH with HCl (see Note 1). Make up to 2 L with distilled water, then add 800 μL of Tween, mix well, and store at room temperature. 2.2

Antibodies Antibody References Species Clone

Supplier

Concentration Antigen (μg/mL) Retrieval

C1q

A0136

Rabbit IgG

Polyclonal Dako

7.90

pH high

C3d

A0063

Rabbit IgG

Polyclonal Dako

6.2

pH low

C4d

DB107

Rabbit IgG

A24-T

CD31

NCLCD311A10

Mouse 1A10 IgG1

DB 4.00 biotech

pH high

Leica

pH high

1.14

The sensibility and specificity of the staining has been evaluated carefully fro these antibodies. If other antibodies that those referenced here are used, the quality of the staining is not guaranteed (see Note 2). 2.3 Common for IHC and Immunofluorescense (IF)

1. Slides with formalin-fixed, paraffin-embedded tumor sections, 3 μm thick. 2. Timer. 3. Oven at 37
C. 4. Glass staining dishes with glass slide rack. 5. Clearene. 6. Ethanol 100%, 90%, 70%, and 50%. 7. Distilled water. 8. Dako PT link or Water bath capable of sustaining 95
C for 30 min. 9. Target retrieval solution pH 9.

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10. Target retrieval solution pH 6. 11. TBS 1. 12. TBS Tween 0.04%. 13. Humidified black chamber with black lid. 14. Oxygenated water 30 volumes (9%). 15. Blocking solution. 16. Antibody diluent. 17. Primary antibody: rabbit polyclonal anti human C1q (Dako), rabbit monoclonal anti human C4d (DB-biotech, Clone A24-T), rabbit polyclonal anti human C3d (Dako), mouse monoclonal anti human CD31 (Leica, Clone AA10). 18. Microscope cover glasses 40 64 mm. 19. Slide scanner. 2.4

Specific for IHC

1. Secondary antibodies: Polymer HRP Rabbit (Peroxidase labelled polymer conjugated to goat anti-rabbit immunoglobulins in TBS 1 buffer containing (see Note 3). 2. AEC (3-amino-9-ethylcarbazole). 3. Hematoxylin (see Note 4). 4. Aqueous based mounting medium. 5. Hot plate.

2.5

Specific for IF

1. Secondary antibodies: (a) Classical immunofluorescence: Goat anti rabbit Cy5, Goat anti mouse Cy3 IgG1. (b) Tyramide immunofluorescence: AF647 tyramide reagent, AF546 tyramide reagent. 2. DAPI (40 ,6-diamidino-2-phenylindole). 3. Mounting medium that preserves fluorescence signal across the entire visible spectrum and causes little or no quenching of the initial signal.

3 3.1

Methods Heating

3.2 Deparaffinization and Rehydration

1. Put the slides in the oven at 37
C for at least 30 min. 1. Place 8 clean glass staining dishes under a chemical fume hood. Fill 3 of them with Clearene solution (see Note 5) in order to recover the slide rack. The next four clean glass staining dishes are filled with ethanol gradient (100%, 90%, 70%, and 50%). The last one is filled with distilled water. Preset timer for 5 min. 2. Place slide(s) in the glass slide rack and put it in the different clean glass staining dishes in order to submerge all tissue

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sections for 5 min in the following order: Clearene #1, Clearene #2, Clearene #3, ethanol 100%, ethanol 90%, ethanol 70%, ethanol 50%, and distilled water. 3.3

Antigen Retrieval

1. Prewarm the water bath containing the antigen retrieval solution (pH high or Low) at 65
C (see Note 6). Put the rack containing the slide in the corresponding bath (High or Low pH). Warm to reach 97
C and leave the slide for 30 min. Let the bath cool to minimum 60
C before remove the slide rack. 2. Rinse the slide for 5 min with the Dako Wash solution (0.05 mol/L Tris–HCl, 0.15 mol/L NaCl, 0.05% Tween 20, pH ¼ 7.6).

4

Staining For all the solutions used, prepare 200 μL of solution by slide.

4.1 IHC Staining: C1q, C4d, and C3d (Figs. 1, 2, and 3)

1. Prepare a black humidified chamber by adding a layer of water. Gently place slides on the plastic rails and put the lid. 2. Prepare a solution of 3% oxygenated water by diluting 1:3 the 9% stock solution with distilled water. 3. Dry the tissue by carefully absorbing the excess of water with absorbent paper towels (see Note 7). 4. Without waiting, add carefully 200 μL of % oxygenated water 3 on each the tissue section for 15 min at room temperature.

Fig. 1 C1q staining on ccRCC sections. Two types of staining are detected; a cytoplasmic (left) and an extracellular as the surface of tumor cells (right)

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Fig. 2 C4d staining on ccRCC sections. Two types of staining are detected; a cytoplasmic reflecting C4 production by tumor cells (left) and an extracellular as the surface of tumor cells reflecting complement activation (right)

Fig. 3 C3d staining on ccRCC sections. Two types of staining are detected; a cytoplasmic reflecting C3 production by tumor cells (left) and an extracellular as the surface of tumor cells reflecting complement activation (right)

5. Put the slide in a glass staining dish containing a slide rack, a bar magnet and filled with TBS Tween 0.04% in order to wash. Repeat this step with new TBS Tween 0.04% bath two times. Remove the slide and dry them as before and put it back to the humidified chamber.

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6. Without waiting, add carefully 3–4 drops of ready-to-use blocking solution and wait for 30 min. 7. During the incubation period, prepare the primary antibodies by diluting the antibody stock solution in the antibody diluent buffer (see Note 8), vortex the solution and stock at 4
C until ready to apply to tissues. 8. At the end of the incubation time, dry the slides as before and put it back to the humidified chamber. 9. Apply 200 μL of the primary antibody and making sure to cover the entire tissue surface. Incubate at room temperature for 30 min. 10. Repeat step 5. 11. Without waiting, add carefully 3–4 drops of ready-to-use rabbit polymer HRP solution and wait for 30 min. 12. Repeat step 5. 13. During the wash step, prepare AEC solution by adding 2 drops of buffer stock solution (approximately 72 μL), 3 drops of AEC stock solution (approximately 90 μL), and 2 drops of hydrogen peroxide solution (approximately 80 μL) and mix well before use. 14. Remove the slide and dry them as before and put it in a white paper towel on the bench. 15. Add 200 μL of AEC solution on each tissue and wait for 30 min. Watch carefully the staining appearance under a microscope (see Note 9). 16. Wash two times in TBS tween 0.04% and one time in distilled water for 5 min each. 17. Dry the slides and put 200 μL of hematoxylin counterstain solution for 1–10 min (see Note 10). 18. Wash the slides in distilled water 5 min two times. 4.2 Classical Double Immunofluorescence Staining: C1q/CD31 (Fig. 4)

1. Perform the steps 1–6 described above to achieve slides blocking. 2. During the incubation period, prepare the mix of the primary antibodies (C1q, CD31) by diluting the antibody stock solution in the antibody diluent buffer (see Note 11), vortex the solution and stock at 4
C until ready to apply to tissues. 3. At the end of the incubation time, dry the slides as before and put it back to the humidified chamber. 4. Apply 200 μL of the primary antibody mix and making sure to cover the entire tissue surface. Incubate at room temperature for 1 h.

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Fig. 4 Double staining of C1q(green)/CD3(red). Some endothelial cells are positives for C1q staining

5. Put the slide in a glass staining dish containing a slide rack, a bar magnet and filled with TBS Tween 0.04% in order to wash. Repeat this step with new TBS Tween 0.04% bath two times. Remove the slide and dry them as before and put it back to the humidified chamber. 6. During the washing steps, prepare the secondary antibody by diluting the secondary goat anti-rabbit Cy5 and goat antimouse Cy3 (see Note 12). 7. Add 200 μL of the secondary antibody solution freshly prepare to the slide in the humidified black chamber. Put the black lid and incubate for 30 min. 8. Repeat step 5. Cover the glass staining dish aluminum to avoid light exposure. 9. During the washing steps, prepare the DAPI solution by diluted DAPI stock solution at 1 μg/mL in distilled water. 10. Add 200 μL of DAPI solution freshly prepare to the slide in the humidified black chamber. Put the black lid and incubate for 5 min. 11. Wash three times in distilled water. Cover the glass staining dish aluminum to avoid light exposure. 4.3 Tyramide Signal Amplification Multiplex Immunofluorescence Staining: C1q/C4d (Fig. 5)

1. Prewarm the water bath containing antigen retrieval solution pH High or Low at 65
C. 2. Perform the steps 1–12 of the immunohistochemistry staining method (see Note 13).

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Fig. 5 Double staining of C1q(green)/C4d(red). C1q deposits colocalized partially with C4d deposits

3. Prepare the first tyramide solution AF546, by first diluted oxygenated water 1:20 in distilled water (solution A). Then, diluted in TBS 1 tyramide stock solution 1:200 and solution A 1:200. 4. Add 200 μL of the tyramide solution freshly prepare to the slide in the humidified black chamber. Put the black lid. 5. Wash three times in TBS tween 0.04%. Cover the glass staining dish aluminum to avoid light exposure. 6. Put the rack containing the slide in the corresponding bath (High or Low pH). Warm to reach 97
C and leave the slide for 10 min. Let the bath cool to minimum 60
C before remove the slide rack. 7. Perform the steps 6–12 of the immunohistochemistry staining method. 8. Prepare the second tyramide solution AF647, by first diluted oxygenated water 1:20 in distilled water (solution A). Then, diluted in TBS 1 tyramide stock solution 1:200 and solution A 1:200. 9. Add 200 μL of the tyramide solution freshly prepare to the slide in the humidified black chamber. Put the black lid. 10. Wash three times in TBS tween 0.04%. Cover the glass staining dish aluminum to avoid light exposure. 11. During the washing steps, prepare the DAPI solution by diluted DAPI stock solution at 1 μg/mL in distilled water.

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12. Add 200 μL of DAPI solution freshly prepare to the slide in the humidified black chamber. Put the black lid and incubate for 5 min. 13. Wash three times in distilled water. Cover the glass staining dish aluminum to avoid light exposure.

5

Mounting 1. If needed, warm the mounting medium in hot water at 150
C on a hot plate. 2. Dry the slide as explained before. 3. Put the 24  40 mm cover glasses (see Note 14). 4. Add one drop of mounting medium in the center of the cover glass. 5. Carefully bring the slide more and more close from the drop of mounting medium until the slides are in contact. Let the mounting medium diffuse by capillarity in the all slide and recover all the tissue. 6. Leave to dry for 1 h.

6

Scan 1. Clean the slide with ethanol. 2. Scan the entire slide at 20 and semiquantify the data (see Note 15), separating the quantification of the cytoplasmic staining and the deposits (see Note 16).

7

Notes 1. Turn on your pH meter, be careful the electrode is very fragile. Take the electrode out of its storage solution, wash it with distilled water and dry it carefully with a tissue. Take your solutions of calibration in our case the pH 7.0 and pH 10.0. Start the measure (by pressing the button “measurement” or “cal” depending on your pH meter), put your electrode in the bottle with the pH of 7, let the pH stabilize (during 1 or 2 min), and set the measure (by pressing the button “measurement” or “cal” depending on your pH meter). Rinse the electrode with distilled water and dry it with a tissue. Place the electrode in the pH 10.0 calibration bottle and start the measurement, once the pH is stabilized (1 or 2 min) set the measure. Before starting the measurement of your buffer, rinse the electrode with distilled water and dry it with a tissue. Put

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the electrode in your buffer and let the pH stabilize for 1 or 2 min. Put a magnetic stick bar in your bottle of buffer and start to mix. Add slowly HCl (37%) to your buffer until you reach the desired pH, 7.4. Rinse the electrode with distilled water, dry it with a tissue, put the electrode in the storage solution, and turn off the pH meter. 2. Of note, we have tested other anti-C4d antibodies (Cell Marque, 04A-16) but no satisfactory staining was detected. 3. This system is based on an HRP labeled polymer which is conjugated with secondary antibodies. The rabbit polymer HRP is an extremely sensitive reagent and, as a result, optimal dilutions of the primary antibody are higher than those used for the traditional peroxidase anti-peroxidase method. This protocol offers an enhanced signal generating system for the detection of antigens present in low concentrations or for low titer primary antibodies. 4. AEC reaction product is soluble in alcohols and other solvent. Use a hematoxylin solution without any solvents. Hematoxylin (Dako, S3301) is good to stain nucleus when a AEC revelation is used. 5. Clearene is a mix of selected terpenes and must be handled cautiously. It replaces xylene and toluene. It is less oily than the others solvents and dry faster without leaving residues. 6. The pH of the antigen retrieval solution depends on the antibody used. For the reagents used in this protocol, for anti-C1q antibody the pH is High, for anti-C4d antibody the pH is High, for anti-C3d antibody the pH is Low. 7. Be very careful when absorbing the excess of water. Take an absorbent paper, make a triangle pointing and circumvent the tissue with precautions. Do not let the slide dry completely because it can cause background. 8. The optimal final concentration for the antibodies are indicated below: (a) anti-C1q antibody (Dako, A0136): 7.9 μg/mL. (b) anti-C4d antibody (DB Biotech, DB107): 4 μg/mL. (c) anti-C3d antibody (Dako, A0063): 6.2 μg/mL. 9. The incubation time for AEC is variable between experiments. Please check, the appearances of the staining on microscope and stop the reaction before 30 min if some background begins to appear. 10. The efficacy of hematoxylin staining decreases in time after opening. Make a regular test to determine the best incubation time to stain the nucleus.

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11. In the case of classical double immunofluorescence staining, the two primary antibodies used must be from different species. In the case of C1q/CD31, C1q is a rabbit polyclonal antibody and CD31 a monoclonal mouse antibody. The two primary antibodies are mixed together in the antibody diluent buffer. The optimal final concentration for the anti-CD31 used in this protocol is 1.15 μg/mL and the pH for the antigen retrieval solution is High. 12. The two secondary antibodies must be coupled with a different fluorochrome and mixed together in the antibody diluent buffer. If possible, FITC should be avoided since it causes a lot of autofluorescence. For C1q/CD31 staining, the 2 secondary antibodies are a goat anti-rabbit Cy5 conjugated and a goat anti-mouse Cy3 conjugated. 13. Tyramide Signal Amplification amplify the fluorescence signal and allow high resolution images and the optimal concentration of the antibodies must be adjusted (see below). (a) Anti-C1q antibody (Dako, A0136): 2 μg/mL. (b) Anti-C4d antibody (DB Biotech, DB107): 2 μg/mL. In the case of tyramide signal amplification multiplex immunofluorescence staining, the two staining must be performed one after another. The two primary antibodies used can be from the same species. In the case of C1q/ C4d, both are rabbit antibodies. 14. The length of the cover glass depends of the size of your tissue. 15. The quantification of the deposits and the cytoplasmic staining has to be done in two separate categories, because it brings different information. If a cohort of patients is studied, it is recommended to quantify the staining on the tumor sections in 3 categories: very low (below 5% of total number of infiltrating or tumor cells respectively for C1q+ or C4d+ and C3d+), low (5–30%), and high (over 30%) stainings. A survival analysis should be carried out for the 3 groups. If two of the groups show similar survival, they should be pooled and the analysis— carried out with only 2 groups. 16. The cytoplasmic staining reflects the production of the protein by the cells, while the membranous one—the deposits. The two are not necessarily present on the same cells. The deposits reflect the activity of the complement cascade.

Acknowledgments This work was supported by a grant from Association pour la Recherche sur le Cancer (ARC); Cancer Research for Personalized Medicine (CARPEM) (to LTR) and La Ligue contre le cancer

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(RS19/75-111 to LTR). This work was also supported by INSERM, University Paris Descartes, Sorbonne University, CARPEM, and the Labex Immuno-Oncology Excellence Program. MVD received a PhD fellowship from ARC. References 1. Roumenina L, Daugan MV, Petitprez F, Saute`sFridman C, Fridman WH (2019) Contextdependent roles of complement in cancer. Nat Rev Cancer 19(12):698–715 2. Merle NS, Noe R, Halbwachs-Mecarelli L, Fremeaux-Bacchi V, Roumenina LT (2015) Complement system part II: role in immunity. Front Immunol 6:257 3. Reis ES, Mastellos DC, Ricklin D, Mantovani A, Lambris JD (2018) Complement in cancer:

untangling an intricate relationship. Nat Rev Immunol 18:5–18 4. Ajona D et al (2013) Investigation of complement activation product C4d as a diagnostic and prognostic biomarker for lung cancer. J Natl Cancer Inst 105:1385–1393 5. Roumenina LT et al (2019) Tumor cells hijack macrophage-produced complement C1q to promote tumor growth. Cancer Immunol Res 7:1091–1105. https://doi.org/10.1158/ 2326-6066.CIR-18-0891

Chapter 19 Analysis of the Ligand Recognition Specificities of Human Ficolins Using Surface Plasmon Resonance Nicole M. Thielens, Evelyne Gout, Monique Lacroix, Jean-Baptiste Reiser, and Christine Gaboriaud Abstract Ficolins are innate immune recognition proteins involved in activation of the lectin complement pathway. These oligomeric lectin-like proteins are assembled from subunits consisting of a collagen-like triple helix and a trimeric fibrinogen-like recognition domain. In humans, three ficolins coexist: they differ in their ligand binding specificities, but share the capacity to associate with proteases through their collagen-like stalks and trigger complement activation. We describe methods to decipher the recognition specificities of ficolins, based on surface plasmon resonance, an optical technique allowing real-time and label-free monitoring of biomolecular interactions. This technique was mainly used to characterize and compare binding of the three recombinant full-length ficolins and of their isolated recognition domains to various immobilized BSA-glycoconjugates, acetylated BSA or biotinylated heparin. The avidity phenomenon that enhances the apparent affinity of interactions between oligomeric lectin-like proteins and the multivalent ligands is also discussed. Key words Surface plasmon resonance, Ficolins, Molecular interactions, Recognition specificity, Neoglycoproteins, Multivalency, Avidity

1

Introduction Ficolins are innate immune recognition proteins that circulate in association with serine proteases involved in activation of the lectin complement pathway (recently reviewed in [1, 2]). These oligomeric proteins are assembled from trimeric subunits comprising an N-terminal collagen-like helix and C-terminal fibrinogen-like (FBG) recognition domains. Three ficolins have been identified in humans, ficolin-1 (formerly called M-ficolin), ficolin-2 (L-ficolin), and ficolin-3 (H-ficolin). Although the three ficolins have different binding specificities mediated by their FBG domains, a common hallmark is their capacity to trigger activation of complement proteases called mannose-binding lectin (MBL)-associated serine proteases (MASPs) that are associated to their collagen-like stalks. In

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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the same way as other initiators of the lectin pathway, such as MBL [3, 4] and collectin CL-LK [5], ficolins are able to sense both pathogen- and apoptotic cells-associated molecular patterns and to trigger immune effector mechanisms, including complement activation and enhancement of phagocytosis of their targets. In contrast to MBL and collectin CL-LK, ficolins do not possess a canonical C-type lectin carbohydrate recognition domain, but have a fibrinogen recognition domain with lectin-like properties [6]. In order to decipher the ligand binding specificities of ficolins, we have developed methods based on detection of real time interactions of recombinant ficolins with BSA glycoconjugates or glycosaminoglycans using surface plasmon resonance (SPR) (Biacore technology). SPR is an optical technique that measures changes in the refractive index on the surface of a thin metal film, arising from concentration changes when molecules bind to or dissociate from the surface. Because mass changes are directly detected, this technique allows monitoring of biomolecular interactions without labeling of the interacting components. One of the interactants (called the ligand) is immobilized on the surface while the interacting partner (the analyte) is injected over the surface in a continuous buffer flow thanks to a microfluidic cartridge. The SPR response, recorded as resonance units (RU) is directly proportional to the change in mass concentration close to the surface and the complex formation/ dissociation is monitored in real time as an increase/decrease in the SPR signal. The sensor surface is composed of a glass plate covered by a thin gold film coated with a biocompatible matrix, in general carboxymethylated dextran in the case of Biacore technology (CM sensor chip). In a typical interaction experiment, one interactant is first immobilized to the dextran CM groups, either directly by covalent amine coupling (involving primary amine groups of lysine residues of proteins) or indirectly through its capture by a previously and covalently coupled molecule (e.g., streptavidin for capture of biotinylated ligands). The analyte is then passed over the surface (association phase of the interaction), followed by buffer (dissociation phase). A pulse injection of an appropriate regeneration solution (selected according to the nature of the interaction) finally allows full removal of the bound analyte without damaging the immobilized compound. Recording of binding curves (sensorgrams) with a series of analyte concentrations and global fitting of the curves to defined binding models allows determination of the kinetic parameters and apparent affinity of the interaction. The use of SPR to study lectin–carbohydrate interaction has been described previously (e.g., see Refs. 7, 8). Particular features are related to the fact that most lectins, including the lectin-like ficolins, are multivalent molecules, assembled from 4 to 6 trimeric subunits (i.e., 12 to 18 polypeptide chains) as observed by electron microscopy imaging of recombinant ficolins [9, 10]. Each

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polypeptide chain contains a binding site in the fibrinogen-like moiety and, even if all binding sites may not be involved at the same time for steric hindrance reasons, in most cases simultaneous engagement of several binding sites will provide avidity, thereby enhancing the apparent affinity of the interaction. This observation is especially valid if the multivalent lectin/ficolin is used in fluid phase, mainly due to rebinding effects to the immobilized glycans during the dissociation phase [11, 12]. In addition, the binding kinetics are markedly influenced by the density of immobilized ligands, as demonstrated in our previous SPR study of the interaction between size-fractionated oligomers of recombinant MBL and surfaces coated with various densities of D-mannose-BSA (Man-BSA) [13]. Taking these limitations into account, we describe methods to compare the ligand binding specificity of the three ficolins using defined BSA glycoconjugate surfaces and recombinant full-length ficolins and their isolated recognition domains. We also provide assays to test the capacity of ficolins to interact with biotinylated heparin and the competitive effect of a sulfated compound for ficolin-2 binding. Finally, we report SPR analysis using the reverse configuration, that is, immobilized ficolins and soluble modified BSA.

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Materials

2.1 SPR Consumables and Buffers

All experiments were performed on a Biacore 3000 SPR system with versions 3.2 of Control and Evaluation softwares (GE Healthcare) (see Note 1). 1. Sensor chips CM5 for Biacore 3000 (GE Healthcare) (see Note 2). 2. Polypropylene (PP) vials (diameter 7 mm, 0.8 mL) and penetrable rubber cups (see Note 3). 3. Borosilicate glass vials (diameter 16 mm, 4 mL). 4. Amine coupling kit (GE Healthcare): 750 mg 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 115 mg N-hydroxysuccinimide (NHS), 10.5 mL 1.0 M ethanolamine–HCl pH 8.5 (see Note 4). 5. Immobilization buffers: 10 mM sodium acetate pH 4.0, 4.2, or 5.0, and 10 mM formate pH 3.0. Dissolve 0.41 g anhydrous sodium acetate or 0.34 g sodium formate in 450 mL distilled water. Adjust the pH to the desired value with acetic acid or formic acid, respectively, before adjusting the volume to 500 mL. Filter the solutions on a 0.22 μm filter and store at 4
C (see Note 5).

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6. Running buffers for immobilization: 10 mM Hepes, 150 mM NaCl, pH 7.4, 0.005% Surfactant P20 (HBS-P, GE Healthcare) or HBS-P containing 3.4 mM EDTA (HBS-EP, GE Healthcare) (see Note 6). 7. Stock solution for conditioning the streptavidin surface: 2 M NaCl in 100 mM NaOH. Dissolve 1 g NaOH pellets and 29.2 g NaCl in 250 mL distilled water. Filter on a 0.22 μm filter and store at 4
C. 8. Stock 0.4 M EDTA solution. Mix 58.44 g EDTA and 25 g of NaOH pellets in 500 mL distilled H2O, which yields a 0.4 M solution with a pH of approximately 7.8. Filter on a 0.22 μm filter and store at 4
C. 9. Running buffer for ficolin binding (HBS-CaP): 20 mM Hepes, 150 mM NaCl, CaCl2 1 mM, pH 7.4, 0.005% Surfactant P20 (see Note 7). Dissolve 2.38 g Hepes and 8.76 g NaCl, in 950 mL distilled water. Add 1 mL of a 1 M CaCl2 stock solution (obtained by dissolving 14.7 g calcium chloride dihydrate in 100 mL distilled water) and adjust the pH to 7.4 before adjusting the volume to 1 L. Filter on a 0.22 μm filter and add 0.5 mL surfactant P20 (stock solution 10%, GE-Healthcare). The binding buffer with 1 mM EDTA is prepared in the same way, except that 2.5 mL of 0.4 M EDTA solution are added instead of CaCl2. Store running buffers at 4
C. 10. Regeneration solutions: 2 M NaCl (11.69 g in 100 mL distilled water); 1 M NaCl, 10 mM EDTA (5.84 g NaCl and 2.5 mL EDTA 0.4 M solution in 100 mL distilled water); 3 M MgCl2 (36 g anhydrous MgSO4 in 100 mL distilled water), 1 M Na2SO4 (14.2 g in 100 mL distilled water), 1 M Na2CH3COO, pH 7.2 (8.2 g in 100 mL distilled water with pH adjusted to 7.2); 0.3 M D-galactose (Gal) (2.7 g in 50 mL HBS-P); and 0.3 M N-acetylgalactosamine (GalNAc) or 0.3 M N-acetylglucosamine (GlcNAc) (3.3 g in 50 mL HBS-P). All solutions are filtered on a 0.22 μm filter and stored at 4
C. 11. Sodium dodecyl sulfate (SDS) 0.5% (BIAdesorb solution 1, GE Healthcare). This stock solution is stored at room temperature, but diluted solutions (prepared in distilled water) are stored at 4
C. 2.2 Immobilized Ligands

1. Bovine serum albumin (BSA) (Sigma-Aldrich).

2.2.1 Protein Ligands

3. BSA glycoconjugates, 14 atom spacer (Dextra Laboratories): Gal-BSA; GalNAc-BSA; GlcNAc-BSA (see Note 8). Stock solutions at 1 mg/mL of BSA, Ac-BSA and BSA glycoconjugates in HBS-P (immobilization buffer) are prepared, aliquoted and stored at 20
C.

2. Acetylated BSA (Ac-BSA) (Sigma-Aldrich).

4. Streptavidin (SA) (Sigma-Aldrich). A stock solution at 2 mg/ mL in HBS-P (immobilization buffer) is prepared, aliquoted and stored at 20
C.

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1. Biotinylated heparin (Heparin-biotin sodium salt, SigmaAldrich) (see Note 9). 2. Sucrose octasulfate sodium salt (SOS) (US Biological). A 100 mM stock solution is prepared by dissolving 1.16 g in 10 mL HPS-P and stored at 4
C. A 8 mM solution is prepared by diluting 80 μL of the stock solution in 1 mL HBS-P.

2.3 Soluble Protein Analytes 2.3.1 Recombinant Ficolin-1

1. Full-length recombinant human ficolin-1 was produced in Schneider S2 Drosophila cells stably transfected with the pMT/Bip/V5-His A vector containing the cDNA of ficolin-1 and purified from the culture supernatants by affinity chromatography on a GlcNAc-agarose column (Sigma-Aldrich), as described by Gout et al. [14]. The concentration of purified ficolin-1 was estimated using an absorbance coefficient (A0.1%, 1 cm) at 280 nm of 1.76 and a protomer Mr value of 32,342, giving an Mr value of 388,000 for full-length ficolin-1, assuming assembly as tetramers of trimeric subunits. 2. A DNA segment encoding the C-terminal fibrinogen (FBG) domain of ficolin-1 (residues 80–297 of the mature human protein) was cloned in frame with the melittin signal peptide of the pNT-Bac baculovirus transfer vector [15] and the recombinant baculovirus was generated using the Bac-toBac™ system (Invitrogen). High Five cells were infected with the recombinant virus and the recombinant protein was purified from the culture supernatant by ion exchange chromatography on a Q-Sepharose Fast Flow column (GE Healthcare), as described by Garlatti et al. [16]. The concentration of the purified trimeric ficolin-1 FBG domain was estimated using an absorbance coefficient (A0.1%, 1 cm) at 280 nm of 2.30 and a mass value of 73,650 Da.

2.3.2 Recombinant Ficolin-2

1. Full-length recombinant human ficolin-2 was produced in CHO K1 cells stably transfected with the pcDNA3.1(+) vector containing the cDNA of ficolin-2 and purified from the culture supernatants by affinity chromatography on N-acetylcysteineSepharose as described previously [9, 17]. The concentration of purified ficolin-2 was estimated using an absorbance coefficient (A0.1%, 1 cm) at 280 nm of 1.76 and an Mr value of 406,300, assuming that the proteins mainly associate as tetramers of trimers (protomer Mr value of 33,860). 2. Recombinant full-length ficolin-2 was produced in S2 insect cells (as described for ficolin-1) and purified in the same way as ficolin-2 produced in CHO cells. 3. The C-terminal FBG domain of ficolin-2 (residues 70–288 of the mature human protein) was produced in baculovirusinfected High Five cells as described for ficolin-1 FBG domain (see Subheading 2.3.1). The recombinant protein was purified

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from the culture supernatant by ion exchange chromatography on a Q-Sepharose Fast Flow column (GE Healthcare), as described by Garlatti et al. [18]. The concentration of the purified trimeric ficolin-2 FBG domain was estimated using an absorbance coefficient (A0.1%, 1 cm) at 280 nm of 2.22 and a mass value of 78,000 Da. 2.3.3 Recombinant Ficolin-3

1. Full-length recombinant human ficolin-3 was produced in CHO K1 cells stably transfected with the pcDNA3.1(+) vector containing the cDNA of ficolin-3 and purified from the culture supernatant by affinity chromatography on acetylated BSA-Sepharose as described by Jacquet et al. [19]. The concentration of the purified ficolin-3 was estimated using an absorbance coefficient (A0.1%, 1 cm) at 280 nm of 1.94 and the molarity estimated using an Mr value of 396,000, assuming that the protein mainly associates as a tetramer of trimers (protomer Mr value of 33,000). 2. The C-terminal FBG domain of ficolin-3 (residues 58–276 of the mature human protein) was produced in baculovirusinfected High Five cells as described for ficolin-1 FBG domain (see Subheading 2.3.1). The recombinant protein was purified from the culture supernatant by ion exchange chromatography on a DEAE-cellulose DE52 column (Whatman) (see Note 10) and an S-Sepharose Fast Flow column (GE Healthcare), as described by Garlatti et al. [18]. The concentration of the purified trimeric ficolin-3 FBG domain was estimated using an absorption coefficient (A0.1%, 1 cm) at 280 nm of 2.36 and a mass value of 78,000 Da.

3

Methods The SPR system preparation and routine maintenance are described in the Biacore 3000 instrument manual. The temperature is set at 25
C.

3.1 Immobilization of BSA Glycoconjugates 3.1.1 Immobilization of the BSA Reference Ligand

1. Dock a CM5 sensor chip and prime the system with the immobilization buffer (HBS-EP). Docking the sensor chip to the four open channels of the microfluidic cartridge surface defines four independent flow cells (20 nL each) that are used for sample injection, either individually or serially. 2. Use flow cell 1 for immobilization of unmodified BSA to serve as a reference cell (see Note 11). Set the flow rate at 10 μL/min. 3. Activate the surface by injecting 80 μL (8 min) of a 1:1 mixture of 0.4 M EDC and 0.1 M NHS, using the Quickinject command. An increase of the baseline level of 200 to 300 RU is observed at the end of injection.

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4. Quickinject 20 μL of BSA diluted at 10 μg/mL in 10 mM sodium acetate pH 4.0. The injection may be stopped or new injection cycles performed to reach the desired immobilization level. 5. Quickinject 80 μL of 1 M ethanolamine, pH 8.5 to block the unreacted NHS-activated carboxylic groups of the dextran. 6. Determine the amount of immobilized ligand as the difference in the baseline level before activation and after blocking of the surface. Injection of 20 μL BSA at 10 μg/mL is expected to yield an immobilization level of 4000 RU. 3.1.2 Immobilization of BSA Glycoconjugates and Acetylated BSA

1. BSA glycoconjugates are immobilized in the same way as BSA, by repeating steps 3–6 of Subheading 3.1.1 for each of the three individual other flow cells. The immobilization levels of BSA glycoconjugates should be kept comparable to that of BSA on the reference cell. Injection of 40 μL GlcNAc-BSA or GalNAc-BSA at 10 μg/mL and of 50 μL Gal-BSA at 10 μg/ mL is expected to yield immobilization levels ranging from 3500 to 4500 RU. 2. Immobilization of Ac-BSA is performed in the same way, except that the ligand is diluted at 25 μg/mL in 10 mM formate pH 3.0 and that 150 μL diluted Ac-BSA are injected (see Note 12). Under these conditions, an immobilization level of 750–900 RU of immobilized Ac-BSA is obtained. The corresponding BSA reference surface is obtained by injecting 7 μL BSA diluted at 10 μg/mL in 10 mM formate pH 3.0.

3.2 Immobilization of Heparin 3.2.1 Immobilization of Streptavidin

1. Dock a CM5 sensor chip and prime the system with HBS-P. 2. Use flow cells 1 and 2 for simultaneous immobilization of streptavidin. Set the flow rate at 5 μL/min. 3. Activate both surfaces by injecting 50 μL (10 min) of a 1:1 mixture of 0.4 M EDC and 0.1 M NHS. An increase of the baseline level of about 150 RU is observed at the end of injection. 4. Inject 50 μL of SA diluted at 200 μg/mL in 10 mM sodium acetate pH 4.2. 5. Inject 50 μL of 1 M ethanolamine, pH 8.5 to block the unreacted NHS-activated carboxylic groups of the dextran. A level of 3000–3500 RU of immobilized SA is expected (see Note 13).

3.2.2 Immobilization of Biotinylated Heparin

1. Prime the system again with HBS-P and set the flow rate at 10 μL/min. Select flow cell 2 (flow cell 1 with immobilized SA will serve as the reference surface). 2. Condition the SA surface with three consecutive 10 μL injections of 1 M NaCl in 50 mM NaOH (obtained by diluting the stock solution 2-times with distilled water).

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3. Inject 20 μL of biotinylated heparin diluted at 10 μg/mL in HBS-P. 4. Wash needle and sample loop using 50% isopropanol in 1 M NaCl and 50 mM NaOH (obtained by diluting the stock solution 2-times with isopropanol) after ligand injection. An immobilization level of 150–200 RU heparin is expected (see Note 14). 3.3 Immobilization of the Three Ficolins

1. Dock a CM5 sensor chip and prime the system with HBS-EP. 2. Immobilize BSA on flow cell 1 as described in Subheading 3.1.1. Injection of 40 μL BSA diluted at 25 μg/mL in sodium acetate pH 4.0 results in about 11,000 RU immobilized. 3. The three ficolins are immobilized in the same way as BSA, by repeating steps 3–6 of Subheading 3.1.1 for each of the three individual other flow cells. Dilutions are performed in 10 mM sodium acetate, pH 5.0 (see Note 15). Injection of 40 μL ficolin-1 at 22 μg/mL, 120 μL ficolin-2 at 30 μg/mL and of 30 μL ficolin-3 at 30 μg/mL is expected to yield immobilization levels of 11,300 RU for ficolin-1 and about 20,000 RU for ficolin-2 and ficolin-3.

3.4 Characterization of the Binding Specificity of Recombinant Human Ficolins

The binding properties of ficolins and the calcium dependence of the interactions have been studied by comparing the signals obtained at identical ficolin concentrations on a set of different immobilized BSA-conjugates. An example is presented with GlcNAc-, Gal-, and GalNAc-BSA. 1. BSA, GlcNAc-BSA, Gal-BSA, and GalNAc-BSA are immobilized on a CM5 sensor chip as described in Subheading 3.1.2). 2. Prime the system in HBS-P containing 1 mM CaCl2 or 1 mM EDTA. 3. Inject 60 μL of the ficolin sample at a flow rate of 20 μL/min followed by a 3-min dissociation phase with running buffer using the Kinject command over the four flow cells, selecting flow cell 1 with immobilized BSA as the reference cell for automatic subtraction of the blank signal. 4. Regenerate the surface by a 30-s injection of a regeneration solution. Depending on the nature of the interaction, the regeneration solution may consist in 0.3 M sugar solution (Gal, GalNAc, or GlcNAc), 1 M NaCl and 10 mM EDTA, or 1 M sodium acetate, pH 7.2. If necessary, the injections of the regeneration solutions can be combined and/or repeated until the signal returns to the baseline level (before injection). 5. Use the BIAevaluation software to process the data by overlaying the binding curves, zeroing the baseline response and aligning the injection start. As shown in Fig. 1, clear differences

Surface Plasmon Resonance to Study Ficolin Specificities

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Fig. 1 Analysis of the interaction of the three recombinant human ficolins with different immobilized BSA glycoconjugates. Sixty microliters of 1.6 nM ficolin-1 (a), 10 nM ficolin-2 (b), and 10 nM ficolin-3 (c) were injected over GlcNAc-, Gal-, and GalNAc-BSA (3500–4000 RU) in HBS-CaP at a flow rate of 20 μL/ min in the presence of 1 mM CaCl2 (solid lines) or 1 mM EDTA (dashed lines). The binding signals shown were obtained by automatic subtraction of the signal over the BSA reference surface. (Adapted from Gout et al. [14])

are observed in the binding properties of the three ficolins. Ficolin-1 binds to BSA acetylated glycoconjugates (GlcNAcand GalNAc-BSA) in a Ca2+-dependent manner (as exemplified for Gal-NAc-BSA), but not to Gal-BSA (Fig. 1a). Ficolin-

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2 binds to GlcNAc-BSA, Gal-BSA, independently of calcium, but not to GalNAc-BSA (Fig. 1b). Ficolin-3 binds to Gal-BSA and the interaction is maintained, although slightly diminished, in the presence of EDTA (Fig. 1c) (see Note 16 for use of a similar protocol with mutated ficolins). 3.5 Kinetic Analysis of Ficolins Binding to Immobilized Ac-BSA 3.5.1 Generation of the Binding Data

The ligand immobilization level for kinetic analysis should be as low as possible (6000 RU.

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10. We recommended heme dilutions (in HBS-EP) to be done immediately before injection. Long-term storage of heme in aqueous buffer may promote formation of aggregates.

Acknowledgments This work was supported by Institut National de la Sante´ et de la Recherche Me´dicale (INSERM, France), and by grant from Fondation ARC pour la recherche sur le cancer, France (PJA 20171206410). References 1. Kuhl T, Imhof D (2014) Regulatory Fe(II/III) heme: the reconstruction of a molecule’s biography. Chembiochem 15:2024–2035 2. Umbreit J (2007) Methemoglobin--it’s not just blue: a concise review. Am J Hematol 82:134–144 3. Dutra FF, Alves LS, Rodrigues D et al (2014) Hemolysis-induced lethality involves inflammasome activation by heme. Proc Natl Acad Sci U S A 111:E4110–E4118 4. Figueiredo RT, Fernandez PL, Mourao-Sa DS et al (2007) Characterization of heme as activator of toll-like receptor 4. J Biol Chem 282:20221–20229 5. Graca-Souza AV, Arruda MA, De Freitas MS et al (2002) Neutrophil activation by heme: implications for inflammatory processes. Blood 99:4160–4165 6. Larsen R, Gozzelino R, Jeney V et al (2010) A central role for free heme in the pathogenesis of severe sepsis. Sci Transl Med 2:51ra71 7. Martins R, Maier J, Gorki AD et al (2016) Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions. Nat Immunol 17:1361–1372 8. Roumenina LT, Rayes J, Lacroix-Desmazes S et al (2016) Heme: modulator of plasma systems in hemolytic diseases. Trends Mol Med 22:200–213 9. Wagener FA, Eggert A, Boerman OC et al (2001) Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase. Blood 98:1802–1811 10. Wagener FA, Volk HD, Willis D et al (2003) Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev 55:551–571 11. Dutra FF, Bozza MT (2014) Heme on innate immunity and inflammation. Front Pharmacol 5:115

12. Ghosh S, Adisa OA, Chappa P et al (2013) Extracellular hemin crisis triggers acute chest syndrome in sickle mice. J Clin Invest 123:4809–4820 13. Soares MP, Bozza MT (2016) Red alert: labile heme is an alarmin. Curr Opin Immunol 38:94–100 14. Belcher JD, Chen C, Nguyen J et al (2014) Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 123:377–390 15. Frimat M, Tabarin F, Dimitrov JD et al (2013) Complement activation by heme as a secondary hit for atypical hemolytic uremic syndrome. Blood 122:282–292 16. Merle NS, Grunenwald A, Rajaratnam H et al (2018) Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles. JCI Insight 3:e96910 17. Merle NS, Paule R, Leon J et al (2019) P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent manner. Proc Natl Acad Sci U S A 116:6280–6285 18. Pawluczkowycz AW, Lindorfer MA, Waitumbi JN et al (2007) Hematin promotes complement alternative pathway-mediated deposition of C3 activation fragments on human erythrocytes: potential implications for the pathogenesis of anemia in malaria. J Immunol 179:5543–5552 19. Dimitrov JD, Roumenina LT, Doltchinkova VR et al (2007) Iron ions and haeme modulate the binding properties of complement subcomponent C1q and of immunoglobulins. Scand J Immunol 65:230–239 20. Roumenina LT, Radanova M, Atanasov BP et al (2011) Heme interacts with c1q and

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inhibits the classical complement pathway. J Biol Chem 286:16459–16469 21. Dimitrov JD, Roumenina LT, Doltchinkova VR et al (2007) Antibodies use heme as a cofactor to extend their pathogen elimination activity and to acquire new effector functions. J Biol Chem 282:26696–26706 22. Mcintyre JA (2004) The appearance and disappearance of antiphospholipid autoantibodies subsequent to oxidation--reduction reactions. Thromb Res 114:579–587 23. Mcintyre JA, Faulk WP (2009) Redox-reactive autoantibodies: biochemistry, characterization, and specificities. Clin Rev Allergy Immunol 37:49–54 24. Dimitrov JD, Planchais C, Scheel T et al (2014) A cryptic polyreactive antibody recognizes distinct clades of HIV-1 glycoprotein 120 by an

identical binding mechanism. J Biol Chem 289:17767–17779 25. Gupta N, De Wispelaere M, Lecerf M et al (2015) Neutralization of Japanese encephalitis virus by heme-induced broadly reactive human monoclonal antibody. Sci Rep 5:16248 26. Lecerf M, Scheel T, Pashov AD et al (2015) Prevalence and gene characteristics of antibodies with cofactor-induced HIV-1 specificity. J Biol Chem 290:5203–5213 27. Kanyavuz A, Marey-Jarossay A, LacroixDesmazes S et al (2019) Breaking the law: unconventional strategies for antibody diversification. Nat Rev Immunol 19:355–368 28. Hadzhieva M, Vassilev TL, Roumenina LT et al (2015) Mechanism and functional implications of the heme-induced binding promiscuity of IgE. Biochemistry 54:2061–2072

Chapter 21 Evaluation of Binding Kinetics and Thermodynamics of Antibody–Antigen Interactions and Interactions Involving Complement Proteins Sofia Rossini and Jordan D. Dimitrov Abstract The study of kinetics and thermodynamics of protein–protein interactions can contribute to assessment of the mechanism of molecular recognition process. These analyses can provide information about conformational changes and noncovalent forces that influence the initial recognition between proteins and stabilization of the complex. Studying these aspects may lead to a better comprehension of functions of proteins in biological environment and can become useful for the rational modification of some interactions by engineering of one of the implicated partners. Real-time biosensor assays based on surface plasmon resonance have been widely applied for the label-free evaluation of protein–protein interactions, allowing their characterization in term of binding affinity and kinetics. In the present chapter, we provide a protocol for the assessment of interactions involving complement proteins or antibodies, the protagonists of the immune system. We reported guidelines and indications concerning the analysis of the experimental data for the estimation of the kinetic parameters and for the evaluation of activation and equilibrium binding thermodynamics. Key words Kinetics, Immunoglobulins

1

Thermodynamics,

Protein–protein

interactions,

Complement,

Introduction Evaluation of kinetic and thermodynamic parameters characterizing protein–protein interactions provides valuable information about the mechanism of the molecular recognition [1–5]. These analyses allow for determination not only of the strength of the formed protein–protein complex (binding affinity) but also of the contribution of different variables to the energy that determines the stability of this complex [6–8]. Thus, kinetic and thermodynamic analyses (i.e., evaluation of changes in enthalpy ΔH, changes in entropy TΔS, and changes in free energy ΔG) may reveal the presence of structural adaptations in the interacting molecules and can contribute to the understanding of the major types of noncovalent

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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contacts, driving the formation of intermolecular complexes [6– 10]. Furthermore, the characterization of molecular mechanism underlying protein–protein interactions may play an important role in generation of high affinity ligands for therapeutic targets or in rational engineering of some therapeutically relevant interactions. The principle technique that measures changes in thermodynamic parameters is isothermal titration calorimetry (ITC), which has been widely used for study of binding mechanism of proteins [11, 12]. This method assesses the direct heat (enthalpy) changes that occur during binding of the interacting molecules in solution, allowing for precise estimation of equilibrium thermodynamic parameters, binding affinity, and stoichiometry. However, ITC is not an appropriate method for evaluation of the binding kinetics and hence the activation thermodynamics of the intermolecular interactions. Other limitations of ITC are that it requires relatively high amounts of proteins and that both interacting partners should be in buffer with identical composition. An alternative of ITC for evaluation of the binding thermodynamics of protein–protein interactions is the use of surface plasmon resonance–based (SPR) biosensor assays [10, 13]. This technique permits label free realtime kinetics and equilibrium measurements of interactions between a surface-immobilized molecule and another molecule in solution (analyte). The advantage of biosensor approach is that it requires only minimal quantity of proteins, for example for immobilization of a sensor surface 100 kDa to concentration of 10 μg/mL and protein with

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molecular weight < 100 kDa to concentration of 2–5 μg/mL in 5 mM maleic acid (pH 4, see Note 1) and inject immediately after activation (step 4.1b) of maximum 10 μL (1 min contact time) of this solution. This step allows the covalent immobilization of the protein via primary amino group from lysine on the preactivated chip surface with a relatively low surface density ( 1000 RU. This can be achieved by prolongation of the contact time of the preactivated sensor surface with the protein solution (>1 min). l

(d) Inject 40 μL (contact time 4 min) of 1 M ethanolamine hydrochloride solution (pH 8.5) to block all activated and nonconjugated carboxyl groups on the chip surface.

l

(e) Repeat the steps b–d for the immobilization of the other proteins on flow channel 3 and flow channel 4, in sequence.

l

(f) To estimate the surface density of the proteins, measure the difference in the baseline between the level obtained after activation with EDC:NHS and the level reached after treatment with ethanolamine.

l

(g) Select flow channel 1 and perform the steps b and d (the step c should be omitted; the flow channel 1 should be used as control).

l

(h) After preparation of all flow channels, set the flow rate at 50 μL/min and run buffer over all flow channels of the sensor chip for at least 2 h. This step is performed to equilibrate the coated surface.

(b) 4.2 Immobilization of proteins on SA sensor chip surface. l (a) The SA sensor chip has precoated surface with streptavidin and thus can be used to immobilize biotinylated proteins by a high affinity capture, allowing a homogeneous orientation on the surface. l

(b) In case of SA sensor chip, the activation of the surface is required. Set the flow rate of the system at 30 μL/min and inject three consecutive times,

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over all flow channels, 30 μL of sodium hydroxide solution (0.05 M NaOH containing 1 M NaCl), allowing each time a contact time of 1 min. l

(c) The immobilization step is performed setting the flow rate at 10 μL/min. The flow channel 1 is used as control surface. On flow channels 2, 3, and 4 different proteins can be immobilized.

l

(d) Select flow channel 2 for immobilization of the protein.

l

(e) Dilute the biotinylated protein to the desired concentration (the concentration may be as low as in the pM range) in the running buffer and inject 10 μL of this solution. The binding between biotin and streptavidin is rapid and 1 min of contact time is enough to achieve the equilibrium binding. The washing of needle after immobilization is recommended. Avoid immobilization of proteins at density that exceeds 500 RU.

l

(f) Repeat the steps d and e for flow channels 3 and 4, in sequence.

l

(g) The flow channel 1 does not require any immobilization; it is ready as control surface.

5. Interaction analyses for evaluation of the binding kinetics. (a) For the binding analysis, set the buffer flow rate at 30 μL/ min. (b) Dilute the sample (analyte, monoclonal antibody, or complement protein) in appropriate running buffer (see Subheading 2.2) to the desired concentration (for moderate affinity interaction we recommend to apply 10 twofold dilutions, starting from 100 nM of protein concentration; for high affinity interaction apply 10 twofold dilutions starting from 5 nM of protein concentration) and inject the diluted protein solution. Inject a volume of 120 μL in order to study the association phase for 4 min and set the dissociation phase for at least 300 s (5 min). (c) To completely remove the analytes from the chip surface, perform a regeneration step injecting 15 μL (30 sec contact time) of the regeneration solution. For the highest concentration of sample, two repetitions of this step may be required. The solution used for regeneration depends on the nature of the interaction and is limited by the stability of the coated protein (see Note 2). Some examples appropriate for antigen–antibody interactions are 5 M MgCl2 (appropriate for CM5 and SA chips), 6 M solution of guanidine hydrochloride (appropriate for CM5 chips)

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and solution of 0.05 N NaOH and 1 M NaCl (appropriate for CM5 chip). (d) Before the next injection, the washing of the needle is recommended. (e) Repeat the steps b–d for all the dilutions of the antibody or complement protein. The samples should be injected from the lowest to the highest concentration, to avoid accumulation on the chip surface. 6. Thermodynamic analyses. Perform the complete kinetics analyses (step 5) at following temperatures: 5, 10, 15, 20, 25, 30, and 35  C by setting each time the temperature at sensor compartment and using the same coated surface (see Note 3). 7. Data analysis for binding kinetics. Perform the analysis of the kinetics parameters by using BIAevaluation software version 4.1 (Biacore) or other software for analysis of SPR biosensor data. After subtraction of the baseline (control surface), fit the data obtained from serial dilutions of sample by global analysis, using the 1:1 Langmuir binding model (or Langmuir binding model with drifting baseline) to evaluate the kinetic rate constants (association and dissociation rate constants) and the binding affinity (see Note 4). For the quality of the fits and their statistical significance, the Chi-square (χ 2) value should be lower than the 10% of the maximal binding response. 8. Data analysis for binding thermodynamics. Use the natural logarithm of the kinetic rate constants (obtained at different temperature) to build the Arrhenius plots, providing the temperature dependence (presented as reciprocal of temperatures in Kelvin degree) of association and dissociation rates (Fig. 1). From the Arrhenius equation (Eq. 1 which is usually used in the logarithmic form, reported in Eq. 2) it is possible to estimate the activation energy of the binding process. K ¼ Ae
Ea=RT ln K ¼


Ea 1 þ ln ðA Þ R T

ð1Þ ð2Þ

The slopes of Arrhenius plot may be obtained with GraphPad Prism software (GraphPad Inc. San Diego, CA), by the application of a linear regression analysis of the experimental kinetic data, and can be used to calculate the activation energy of association and dissociation phases, through the following equation: E a ¼
slope  R

ð3Þ

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ARRHENIUS PLOT Intercept: lnA

16

Slope: - Ea/R

lnk 15 14 13

0.00330 0.00345

0.00360

1/T Fig. 1 Arrhenius plot. The plot depicts the natural logarithm of the rate constants (association and dissociation) versus the reciprocal value of the interaction temperature (in Kelvin degrees). Note that the values of the natural logarithm of the dissociation rate constant would have negative value. The slope of the linear regression line defines the activation energy of the binding process, which is used for determination of the activation thermodynamics of association or dissociation phases

where the slope is ∂[ln(ka/d)]/∂(1/T) and Ea is the activation energy. The activation energy provides information about the activation thermodynamics of association and dissociation processes. By the application of Eyring approach, the evaluation of activation thermodynamic parameters is possible Eqs. (4–6). ΔH 6¼ ¼ E a
RT




ln ka=d =T ¼
ΔH 6¼ =RT þ ΔS 6¼ =R þ ln k0 =h ΔG 6¼ ¼ ΔH 6¼ þ T ΔS 6¼




ð4Þ ð5Þ ð6Þ

where T is the reference temperature expressed in Kelvin (298.15 K), k0 is the Boltzmann’s constant, h is the Planck’s constant, and R is the universal gas constant (8.3144 J mol
1 K
1). In Eq. (5) the kinetic rate constant of association (ka) or of dissociation (kd), estimated at reference temperature (25  C or 298.15 K) should be provided. Finally, the equilibrium thermodynamics can be estimated by the use of following relations Eqs. (7–9): 

ΔH ¼ ΔH 6¼ assoc
ΔH 6¼ dissoc 

T ΔS ¼ T ΔS 6¼ assoc
T ΔS 6¼ dissoc

ð7Þ ð8Þ

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VAN ‘T HOFF PLOT Intercept: ΔS/R

22

Slope: - ΔH/R

21

lnKA 20 19 18

0.00330 0.00345

0.00360

1/T Fig. 2 Van’t Hoff’s plot. The plot represents the natural logarithm of equilibrium association constant versus the reciprocal value of the interaction temperature (in Kelvin degrees). The slope of the plot provides information about the changes in the equilibrium enthalpy. The intercept of the liner regression on the ordinate allows the calculation of the changes in entropy at equilibrium 

ΔG ¼ ΔG 6¼ assoc
ΔG 6¼ dissoc

ð9Þ

The equilibrium thermodynamics can be also studied by another approach. Thus, the equilibrium association constants calculated in each temperature can be used to build the Van’t Hoff plot (Fig. 2). By using the Van’t Hoff’s Eq. (10) the equilibrium thermodynamic parameters can be obtained. 



ln K A ¼
ΔH =RT þ ΔS =RT 



ΔG ¼ ΔH
T ΔS



ð10Þ ð11Þ

In the Eq. (10), T is the reference temperature expressed in Kelvin (298.15 K) and R is the universal gas constant (8.3144 J mol
1 K
1). The slope of the Van’t Hoff’s plot can be used directly for estimation of the enthalpy changes (heat released or acquired by the system) during the binding process. The intercept of the plot on the ordinate determines the equilibrium changes in entropy. By using the Eq. (11) the changes in the free energy can be estimated.

4

Notes 1. The referred pH value of maleic acid solution is appropriate for immobilization of gp120, human IgG, and human IgM as well as C-reactive protein. For other proteins solutions with higher

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pH can be applied. Note that the pH value of this solution should be always at least two points lower than the isoelectric point of the protein. 2. In case of SA sensor chip, avoid the use of basic solutions for regeneration. They may promote the leaching of the biotinylated ligand on the sensor surface and the contamination of other flow channels. 3. After subsequent injections, the protein immobilized on the chip surface may be degraded and may lose the binding capacity. In case of CM5 chip surface, the immobilization is covalent and irreversible and the problem can be solved using a new coating procedure on novel chip. In case of SA sensor chip the immobilization is non covalent and the removal of the protein from the chip surface is possible by a brief injection (30 sec contact time) of an aggressive solution (solution of 6M urea). 4. Use the Langmuir binding model only if the monovalence of the interaction is certain (e.g., interaction of IgG with sparsely immobilized antigen, i.e., 100–500 RU). In case of multivalent interactions such as in case of the complement protein C1q with its ligand molecules immobilized at high density, appropriate multivalence kinetic model should be applied.

Acknowledgments This work was supported by Institut National de la Sante´ et de la Recherche Me´dicale (INSERM, France), Centre National de la Recherche Scientifique (CNRS, France). S.R. was supported by Erasmus scholarship (European program Erasmus+ Traineeship, University of Perugia, Italy. References 1. Amzel LM (2000) Calculation of entropy changes in biological processes: folding, binding, and oligomerization. Methods Enzymol 323:167–177 2. Amzel LM, Siebert X, Armstrong A et al (2005) Thermodynamic calculations in biological systems. Biophys Chem 117:239–254 3. Chodera JD, Mobley DL (2013) Entropyenthalpy compensation: role and ramifications in biomolecular ligand recognition and design. Annu Rev Biophys 42:121–142 4. Schreiber G (2002) Kinetic studies of proteinprotein interactions. Curr Opin Struct Biol 12:41–47

5. Schreiber G, Haran G, Zhou HX (2009) Fundamental aspects of protein-protein association kinetics. Chem Rev 109:839–860 6. Chothia C, Janin J (1975) Principles of protein-protein recognition. Nature 256:705–708 7. Janin J (1995) Principles of protein–protein recognition from structure to thermodynamics. Biochimie 77:497–505 8. Ross PD, Subramanian S (1981) Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20:3096–3102 9. Mariuzza RA, Poljak RJ, Schwarz FP (1994) The energetics of antigen-antibody binding. Res Immunol 145:70–72

Evaluation of Binding Kinetics and Thermodynamics 10. Myszka DG (2000) Kinetic, equilibrium, and thermodynamic analysis of macromolecular interactions with BIACORE. Methods Enzymol 323:325–340 11. Chaires JB (2008) Calorimetry and thermodynamics in drug design. Annu Rev Biophys 37:135–151 12. Pierce MM, Raman CS, Nall BT (1999) Isothermal titration calorimetry of protein–protein interactions. Methods 19:213–221 13. Lipschultz CA, Yee A, Mohan S et al (2002) Temperature differentially affects encounter and docking thermodynamics of antibody-antigen association. J Mol Recognit 15:44–52 14. Manivel V, Bayiroglu F, Siddiqui Z et al (2002) The primary antibody repertoire represents a linked network of degenerate antigen specificities. J Immunol 169:888–897 15. Manivel V, Sahoo NC, Salunke DM et al (2000) Maturation of an antibody response is governed by modulations in flexibility of the antigen-combining site. Immunity 13:611–620 16. Willcox BE, Gao GF, Wyer JR et al (1999) TCR binding to peptide-MHC stabilizes a

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flexible recognition interface. Immunity 10:357–365 17. Maenaka K, Van Der Merwe PA, Stuart DI et al (2001) The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties. J Biol Chem 276:44898–44904 18. Hadzhieva M, Pashov AD, Kaveri S et al (2017) Impact of antigen density on the binding mechanism of IgG antibodies. Sci Rep 7:3767 19. Dimitrov JD, Planchais C, Scheel T et al (2014) A cryptic polyreactive antibody recognizes distinct clades of HIV-1 glycoprotein 120 by an identical binding mechanism. J Biol Chem 289:17767–17779 20. Prigent J, Jarossay A, Planchais C et al (2018) Conformational plasticity in broadly neutralizing HIV-1 antibodies triggers Polyreactivity. Cell Rep 23:2568–2581 21. Roumenina LT, Radanova M, Atanasov BP et al (2011) Heme interacts with c1q and inhibits the classical complement pathway. J Biol Chem 286:16459–16469

Chapter 22 Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications Alessandra Zarantonello, Sofia Mortensen, Nick S. Laursen, and Gregers R. Andersen Abstract Activated complement component C4 (C4b) is the nonenzymatic component of the classical pathway (CP) convertases of the complement system. Preparation of C4 and C4b samples suitable for structural biology studies is challenging due to low yields and complexity of recombinant C4 production protocols reported so far and heterogeneity of C4 in native sources. Here we present a purification protocol for human C4 and describe sample preparation methods for structural investigation of C4 and its complexes by crystallography, small angle X-ray scattering, and electron microscopy. Key words Complement C4, Ion exchange, Gel filtration, X-ray crystallography, Small angle X-ray scattering, Negative stain electron microscopy

1

Introduction

1.1 Overall Structure of Complement C4

The main locus of synthesis of C4 is in the liver [1], but the protein is also locally produced in the brain, reviewed in [2]. The fully mature 195 kDa C4 protein is structurally complex and consists of three chains α, β and γ linked by disulfide bridges. The C4 α chain spans the C4a, the C-terminal half of the MG (macroglobulin) 6, MG7, CUB (complement C1r/C1s, Uegf, Bmp1) and TE (thioester) domains and the first β-strand in the MG8 domain. The C4 β chain is formed by the MG1-5 domains and the N-terminal part of the MG6 domain. The remaining part of the MG8 domain and the C345c (C-terminal domain shared by complement C3, C4 and C5) domain constitute the γ chain [3–5]. There are five glycosylation sites on C4 [6] and five functionally important regions for the early events of the classical pathway (CP) complement cascade: (i) the surface interacting with the C4 cleaving proteases MASP2 and C1s, formed by a sulfotyrosine containing region in the MG8 domain and a positively charged surface area in the C4 C345c

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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domain [3], (ii) the internal thioester cleaved by nucleophiles in nascent C4b [7], (iii) the acidic residues forming the interaction surface with C2 close to the N-terminal of the α chain, (iv) the C-terminus of the γ chain coordinating the Mg2+ ion in the metal ion dependent adhesion site (MIDAS) present in the von Willebrand type A domain of C2 [8, 9], and (v) the binding site for regulators of complement activity C4BP, CR1, MCP, DAF, and CSMD1 [10, 11]. Activation of C4 occurs through the CP and the lectin pathway (LP). In the CP, binding of the C1 complex to an activator triggers C1r and C1s activation [12]. Likewise in the LP, association of MBL/ficolin/CL-LK MASP-1/2 complexes with specific glycan patterns leads to activation of MASP-1 and MASP-2 [13]. Cleavage of C4 by MASP-2 or C1s results in the release of the 10 kDa C4a and initiates a conformational change in nascent C4b leading to exposure of the internal thioester [4] which can react with a nucleophile, thereby opsonizing the activator surface with the 185 kDa C4b [7]. Removal of C4a from the C4 α chain leads to the formation of the α0 chain in C4b. Human complement C4 is the most heterogeneous protein of the complement system, with the two different human isotypes C4A and C4B. The two isotypes differ by four residues located within a stretch of six positions in the thioester domain. Activated C4A (1120PCPVLD1125) preferentially reacts with amine nucleophiles coming from proteins in, for example, immune complexes. Activated C4B (1120LSPVIH1125) reacts more readily with hydroxyl nucleophiles, therefore binding more efficiently to carbohydrates [7, 14]. Recent studies have connected high expression levels of the C4A isotype with an increased risk of developing schizophrenia [15]. 1.2 Convertase Assembly and Regulation of C4b

The CP C3 proconvertase C4b2, formed when C4b binds the C2 zymogen, is activated through cleavage of C2 by C1s, MASP-1, or MASP-2 to yield C4b2a, the CP C3 convertase which may cleave C3. The resulting C3b molecules will further opsonize the activator surface after undergoing a conformational change analogous to that occurring in nascent C4b [7]. Complement deposition is tightly regulated on the surface of host cells, expressing proteins part of the family of regulators of complement activity (RCA). These proteins can act as cofactors (C4BP, MCP, CR1, and CSMD1) for the protease factor I (FI) in the degradation of C4b to the fragments iC4b, C4c, and C4d devoid of the ability to form convertases. Additionally, CR1 and DAF function as competitors of C2a thereby exerting decay acceleration activity on the CP convertase [10, 11]. C3b deposition is regulated through analogous mechanisms and recent structural knowledge established the structural basis for the interaction between members of the RCA family and C3b [16], C4b-regulator contacts are likely to resemble those observed in C3b-regulator complexes.

Purification of Human C4 for Structural Studies

1.3 Sample Preparation for Structural Studies Involving C4 and C4b

2 2.1

251

A deep mechanistic understanding of C4 activation, C4b function in the CP convertase and the mechanism of C4b regulation requires structural data and atomic models. Such models may also form the basis for the development of therapeutic agents specifically controlling complement C4. In this chapter we present a robust protocol for C4 purification from human plasma, yielding a sample suitable for structural analysis, a task which is difficult to accomplish due to the natural heterogeneity of C4 and the low yields and incomplete processing of recombinant C4. We also outline the final steps of sample preparation prior to collection of X-ray diffraction and small angle X-ray scattering data resulting in atomic models as already presented by us [3, 4, 17]. Finally, we present in detail an example of negative stain electron microscopy where the location of single domain antibodies (nanobodies) on the proconvertase C4b2 is determined.

Materials C4 Purification

1. Human citrated outdated plasma from an individual donor, one portion amounts to 330 mL, from local blood bank. The resistivity of water (milliQ) used for buffer preparation and dilutions is higher than 18 MΩ cm. 2. Buffer 1 (see Note 1): 10 mM Tris–HCl pH ¼ 7.5, 100 mM NaCl, 50 mM ε-amino-caproic acid, 5 mM EDTA, 0.5 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM BZA (benzamidine). For 1 L add 10 mL Tris–HCl 1 M stock, 20 mL NaCl 5 M stock, 50 mL ε-amino-caproic acid 1 M stock, 10 mL EDTA 500 mM stock, 5 mL PMSF 100 mM stock, 1 mL BZA 1 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L and filter through 0.22 μm filter. 3. Buffer 2: 20 mM Tris–HCl pH ¼ 7.5, 200 mM NaCl, 0.5 mM PMSF, 1 mM BZA. For 1 L add 20 mL Tris–HCl 1 M stock, 40 mL NaCl 5 M stock, 5 mL PMSF 100 mM stock, 1 mL BZA 1 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L and filter through 0.22 μm filter. 4. Buffer 3: 20 mM Tris–HCl pH ¼ 7.5, 100 mM NaCl, 100 μM PMSF, 100 μM BZA. For 1 L add 20 mL Tris–HCl 1 M stock, 20 mL NaCl 5 M stock, 1 mL PMSF 100 mM stock, 100 μL BZA 1 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter. 5. Buffer 4: 20 mM Tris–HCl pH ¼ 7.5, 200 mM NaCl. For 1 L add 20 mL Tris–HCl 1 M stock, 40 mL NaCl 5 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter.

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6. Buffer 5: 20 mM HEPES–NaOH pH ¼ 7.5, 200 mM NaCl. For 1 L add 20 mL HEPES–NaOH 1 M stock, 40 mL NaCl 5 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter. 7. Buffer 6: 20 mM HEPES–NaOH pH ¼ 7.5, 100 mM NaCl. For 1 L add 20 mL HEPES–NaOH 1 M stock, 20 mL NaCl 5 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter. 8. Buffer 7: 20 mM HEPES–NaOH pH ¼ 7.5, 100 mM NaCl, 2 mM MgCl2. For 1 L add 20 mL HEPES–NaOH 1 M stock, 20 mL NaCl 5 M stock, 2 mL MgCl2 stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter. 9. 50% w/w PEG6000 solution in milliQ water (1 kg PEG6000/ 1 L water). Dissolve 500 g of PEG6000 in 500 mL milliQ water while stirring. Store at 4  C. 10. Resins: Q Sepharose FF, Q Sepharose HP, Source 15Q 9 mL, Mono Q 9 mL. All columns and resins are from GE Healthcare. 2.2

C4b Generation

1. 1 M Tris–HCl pH ¼ 8.8 (see Note 1e). 2. 500 mM glycine (see Note 1k). 3. 100 mM iodoacetamide (see Note 1l). 4. C1s enzyme, Complement Technology, Inc. 5. C1 inhibitor (C1 INH), Complement Technology, Inc.

2.3 C4b Deglycosylation for Crystallization and Sample Preparation for X-Ray Crystallography Data Collection

1. GST-tagged PNGaseF. 2. GST-tagged EndoF1. 3. 1 mL GSTrap FF column (GE Healthcare). 4. Superdex200 Increase 10/300 GL Column (GE Healthcare). 5. Buffer 8: 20 mM HEPES–NaOH pH ¼ 7.5, 150 mM NaCl. For 1 L add 20 mL HEPES–NaOH 1 M stock, 30 mL NaCl 5 M stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter. 6. SWISSCI MRC 2 wells crystallization plates. 7. Oryx robot (Douglas Instruments).

2.4 C4b Deglycosylation and Sample Preparation for SAXS Data Collection

1. GST-tagged PNGaseF. 2. GST-tagged EndoF1. 3. MBP-tagged EndoF2. 4. MBP-tagged EndoF3. 5. GSTrap FF 1 mL column.

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6. MBPTrap HP 5 mL column (GE Healthcare). 7. Superdex200 Increase 10/300 GL column. 2.5 Sample Preparation for Negative Stain Electron Microscopy and Data Collection

1. Superdex200 Increase 10/300 GL column. 2. Buffer 9: 20 mM HEPES–NaOH pH ¼ 7.5, 150 mM NaCl, 3 mM MgCl2. For 1 L add 20 mL HEPES–NaOH 1 M stock, 30 mL NaCl 5 M stock, 3 mL MgCl2 stock to 850 mL milliQ water. Adjust the pH to 7.5, fill to 1 L with milliQ water and filter through 0.22 μm filter. 3. Glow discharged GC400 carbon-coated copper grid (Gilder). 4. Tecnai T12 G2 transmission electron microscope operating at 120 kV equipped with a Teitz TemCam-F416 detector (TVIPS). Software: Leginon, DoG Picker (Appion), EMAN2, RELION 2.1 beta, Chimera v11.1.2.

3 3.1

Methods C4 Purification

1. All purifications step are carried out at 4  C or on ice (see Note 2). 2. Outdated human plasma from a single donor is quickly thawed under tap water (see Notes 3 and 4). 3. BZA (to 10 mM), PMSF (to 1 mM), and PTI (pancreatic trypsin inhibitor) (to 3 μg/mL) are immediately added. 4. 250 mM BaCl2 is added by slow dripping to a final concentration of 60 mM. 5. 250 mM Na3C3H5O(COO)3 (trisodium citrate) is added to a final concentration of 25 mM. 6. The resulting precipitate is pelleted by centrifugation at 6000  g force for 20 min. 7. The supernatant is loaded on a 200 mL Q Sepharose FF column (4  15 cm) equilibrated in buffer 1 using a peristaltic pump at 5 mL/min (see Note 5). 8. The column is washed until baseline absorbance at 20 mL/ min. 9. The elution is performed with a 1200 mL linear NaCl gradient from 100 to 600 mM NaCl at 20 mL/min while collecting 50 mL fractions manually. 10. The resulting fractions are analyzed by reducing SDS-PAGE (see Note 6) and those containing C4 are pooled together as shown in Fig. 1. The 50% w/v PEG6000 solution is slowly added by dripping to a final concentration of 12% w/w (see Note 7). The precipitate is pelleted at 4000  g force for 30 min and the pellet is resuspended in 100 mL buffer 2 (see Note 8).

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Fig. 1 Fast capture of human C4 by anion exchange chromatography. (a) Chromatogram of the elution from Q Sepharose FF 200 mL manually redrawn in Adobe Illustrator from a pen-writer chromatogram. (b). Reducing SDS-PAGE analysis of 15 μL taken from fractions 8–22. The pooled fractions 13–20 are underlined in the chromatogram

11. The resuspended sample is filtered through a 0.45 μm syringe filter and then loaded at 1 mL/min on a Q Sepharose HP 50 mL (2  16 cm) column equilibrated in buffer 2 using a peristaltic pump (see Note 5). The column is washed with buffer 2 until baseline and eluted with a 600 mL linear NaCl gradient from 200 to 800 mM NaCl while collecting 8 mL fractions at 2 mL/min (Fig. 2).

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Fig. 2 Purification on Q Sepharose HP. (a) Chromatogram of the elution from the 50 mL Q Sepharose HP column redrawn from a pen-writer chromatogram. (b) Reducing SDS-PAGE analysis of 15 μL from fractions 28–36 (underlined in chromatogram) of elution from the Q Sepharose HP column

12. The fractions containing C4 are pooled after a reducing SDS-PAGE analysis and diluted 1:3 with water. The diluted sample is loaded on Source 15Q 9 mL column equilibrated in buffer 3 (see Note 9). 13. The column is washed until baseline with buffer 3 supplemented with 200 mM NaCl and eluted with a 100 mL linear NaCl gradient from 200 to 400 mM NaCl at 1.5 mL/min while collecting 4 mL fractions (Fig. 3). The C4 containing fractions are pooled after reducing SDS-PAGE analysis. 14. The C4 pool from the Source 15Q column is diluted 1:3 with water. The sample is loaded on a Mono Q 9 mL column (see Note 10) equilibrated in buffer 4 and the column is eluted with a 100 mL linear NaCl gradient from 200 to 700 mM while collecting 4 mL fractions at 1.5 mL/min (Fig. 4).

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Fig. 3 Purification on Source 15Q. (a) Chromatogram of the elution from the Source 15Q 9 mL column. (b) Reducing SDS-PAGE analysis of 5 μL taken from fractions 8–13 (underlined) in the chromatogram

Fig. 4 Final purification of human C4 on Mono Q. (a) Chromatogram of the elution from the Mono Q 9 mL column. (b) Reducing SDS-PAGE analysis of 5 μL taken from each of fractions 8–14 (underlined) in the chromatogram

15. The fractions containing C4 are pooled after reducing SDS-PAGE analysis. The protein concentration is determined by measuring the UV absorbance at 280 nm and using the molar extinction coefficient ε ¼ 189,230 M1 cm1. Purified C4 may at this stage be stored at 80  C after aliquoting in 1 mL portions and flash freezing in liquid nitrogen. The C4 yield is typically 75 mg/L of thawed plasma. 3.2

C4b Generation

1. The sample is supplemented with 30 mM glycine, 50 mM Tris– HCl pH ¼ 8.8, and 10 mM IAA (see Note 11). C1s is added at 0.1% w/w ratio and the C4 cleavage is carried out for 16 h at 37  C (Fig. 5a).

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Fig. 5 C4 cleavage with C1s and deglycosylation. (a) SDS-PAGE analysis of C4b generated by C1s digestion of C4. Notice the appearance of the α0 chain below the 85 kDa marker and the almost complete degradation of the α chain just below the 100 kDa marker. (b) C4b deglycosylation prior to crystallization, using PNGaseF and EndoF1, (adapted from [4]). C4b before and after incubation with the endoglycosidases is shown for comparison. (c) SDS-PAGE analysis after the final purification of deglycosylated C4b for SAXS analysis after treatment with PNGaseF, EndoF1, EndoF2, and EndoF3 as described [17]

2. After cleavage, C1 INH is added to the sample at 1% w/w ratio to C4 and the sample is incubated on ice for 1 h. 3. The sample is loaded on a 9 mL Source 15Q column equilibrated in buffer 5 and eluted with an 80 mL linear NaCl gradient from 200 to 400 mM to remove contaminants. 4. The fractions containing C4b are pooled after SDS-PAGE analysis and dialyzed against buffer 6. 3.3 C4b Deglycosylation for Crystallization and Sample Preparation for X-Ray Crystallography Data Collection

1. Bacterially expressed PNGaseF and EndoF1 [4, 17] are added at a 1:10 w/w ratio to C4b. 2. The deglycosylation reaction is carried out for 37 h at 30  C. 3. The endoglycosidases are removed by affinity chromatography purification on a GSTtrap column according to manufacturer’s instructions (Fig. 5b). 4. The deglycosylated C4b sample is purified by gel filtration chromatography on a Superdex200 Increase 10/300 GL column equilibrated in buffer 8. 5. The fractions of interest are pooled after reducing SDS-PAGE analysis and the sample is concentrated using 100 kDa MWCO membrane 0.5 mL centrifugal filters. 6. At concentrations in the 4–5 mg/mL range, the C4b protein sample is mixed with a reservoir solution containing 7.5% (w/v) PEG8000, 0.4 M MgCl2, and 0.1 M Tris–HCl pH ¼ 7 and a crystal seed stock solution in a SWISSCI MRC

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96 well 2-drop vapor diffusion plate, to which 50 μL reservoir is pipetted in each well. 7. Crystallization drops of size of 300–600 nL are dispensed with the Oryx robot at a 1:1:0.5 ratio of C4b solution : reservoir solution : crystal seed stock. 8. The plates are incubated at 19  C. 9. The resulting crystals are cryoprotected by soaking them sequentially for 5–10 s in reservoir solution, supplemented with 10, 20 and 30% (v/v) PEG400, prior to mounting in loops and flash cooling in liquid nitrogen for data collection. 3.4 C4b Deglycosylation and Sample Preparation for SAXS Data Collection

1. Purified C4b is added Tris–HCl pH ¼ 7.0 to a final concentration of 50 mM and 10% (w/w to C4b) bacterially expressed GST-tagged Endo F1, GST-tagged PNGase F, MBP-tagged Endo F2, and MBP-tagged Endo F3 [4, 17]. 2. Deglycosylation reaction is carried out at 30  C for 24 h. 3. The endoglycosidases are removed by affinity chromatography on GSTtrap FF and MBPtrap HP columns according to manufacturer’s instructions. 4. The flow through containing deglycosylated C4b (Fig. 5c) is subjected to size exclusion chromatography on a Superdex 200 Increase 30/100 GL column equilibrated in buffer 7. 5. The relevant fractions are pooled after reducing SDS-PAGE analysis and the C4b sample is concentrated using 100 kDa MWCO membrane centrifugal filters to protein concentrations of 10, 5, and 2.5 mg/mL. 6. The dilution series of deglycosylated C4b can be used for SAXS data acquisition. Buffer 7 is used for background subtraction of the SAXS data.

3.5 Sample Preparation for Negative Stain Electron Microscopy and Data Collection

1. For negative stain electron microscopy (NSEM) experiments, the sample is prepared by mixing C4b:C2:Nanobody(Nb)5: Nb10 at 1:1.1:5:5 molar ratio (see Note 12). The complex is incubated at 4  C for 5 min before injection on a Superdex200 Increase 10/300 GL column, equilibrated in buffer 9. The eluted fractions are analyzed by reducing SDS-PAGE and the peak fraction (21) is used for sample preparation for the following NSEM experiment (Fig. 6). 2. Fraction 21 is diluted to 20 μg/mL in buffer 9 and applied to a glow-discharged carbon-coated copper GC400 grid for 5 s. After application, the sample is blotted and stained with 3 μL of 2% w/v uranyl formate by two sequential rounds of staining and immediately blotting, followed by a 1 min stain and blot before the grid is air-dried (Fig. 7).

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Fig. 6 Purification of a proconvertase complex for electron microscopy. (a) Chromatogram after purification on a 24 mL Superdex200 increase of C4b:C2:Nb5:Nb10 ¼ 1:1.1:5:5. (b) SDS-PAGE analysis of the fractions underlined in the chromatogram

Fig. 7 Workflow of NSEM grid preparation. (a) 3 μL of sample are applied to the glow discharged GC400 grid. (b) The grid is incubated for 5 s before blotting the liquid away with a Whatman filter paper. (c, d) 3 μL of uranyl formate stain are added on the grid and immediately blotted away on Whatman filter paper, the wash is performed twice, followed by application of 3 μL stain and incubation of the grid for 1 min upside down, to avoid deposition of uranyl formate crystals on the surface. After 1 min the excess liquid is blotted away. Finally, the grid is left to air-dry before data collection

3. The micrographs are recorded automatically using Leginon on a Tecnai T12 G2 transmission electron microscope operating at 120 kV equipped with a Teitz TemCam-F416 detector (TVIPS). The defocus range is 0.7 to 1.7 μm, exposure time 750 ms and magnification 67,000, yielding a pixel size ˚. of 3.15 A 4. The data are processed without performing contrast transfer function (CTF) correction. The particles are automatically picked by DoG Picker (Appion) and extracted with a box size of 80x80 pixels, using 2 times binning of the data in EMAN2 [18]. 5. The particles used for 3D classification are selected after reference-free two-dimensional (2D) classification in RELION ˚ . The reference density 2.1 beta [19], using a mask of 350 A

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Fig. 8 Three-dimensional reconstruction of the proconvertase complex obtained by negative stain electron microscopy. The reconstruction of C4b2:Nb5:Nb10 and a docked model of C4b2 are shown in the front view (a) and the back view (b). The star shaped labels mark densities containing the putative position of the two nanobodies

map in the 3D reconstruction is generated from the SEC-SAXS ˚ . Fitting of PDB model of C4b2 [17] low-pass filtered to 60 A the PDB model into the 3D EM density is performed in Chimera (Fig. 8).

4

Notes 1. All stock solutions are prepared according to the following instructions. (a) 250 mM BaCl2: add 200 mL milliQ water to a 250 mL graduated cylinder. Weigh 15.3 g of BaCl2·2H2O and transfer to the cylinder. Dissolve by stirring and make up to 250 mL with milliQ water. (b) 250 mM Na3C3H5O(COO)3 (trisodium citrate): add 200 mL milliQ water to a 250 mL graduated cylinder. Weigh 18.4 g of Na3C3H5O(COO)3·2H2O and transfer to the cylinder. Dissolve by stirring and make up to 250 mL with milliQ water. (c) 1 M Tris–HCl pH ¼ 7.0: add 500 mL milliQ water to a 1 L graduated cylinder. Weigh 121.14 g of Tris (tris (hydroxymethyl)aminomethane) and transfer to the cylinder while stirring. Adjust the pH to 7 using concentrated HCl. Make up to 1 L with milliQ water and filter through 0.22 μm filter. (d) 1 M Tris–HCl pH ¼ 7.5: add 500 mL milliQ water to a 1 L graduated cylinder. Weigh 121.14 g of Tris (tris

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(hydroxymethyl)aminomethane) and transfer to the cylinder while stirring. Adjust the pH to 7.5 using concentrated HCl. Make up to 1 L with milliQ water and filter through 0.22 μm filter. (e) 1 M Tris–HCl pH ¼ 8.8: add 500 mL milliQ water to a 1 L graduated cylinder. Weigh 121.14 g of Tris (tris (hydroxymethyl)aminomethane) and transfer to the cylinder while stirring. Adjust the pH to 8.8 using concentrated HCl. Make up to 1 L with milliQ water and filter through 0.22 μm filter. (f) 5 M NaCl: add 500 mL milliQ water to a 1 L graduated cylinder. Weigh 292.2 g of NaCl and transfer to the cylinder while stirring. Make up to 1 L with milliQ water and wait until the salt is completely dissolved. Filter through 0.22 μm filter. (g) 1 M ε-amino-caproic acid: add 500 mL milliQ water to a 1 L graduated cylinder. Weigh 131.2 g ε-Amino-Caproic Acid and transfer to the cylinder. Make up to 1 L and filter through 0.22 μm filter. (h) 0.5 M EDTA–NaOH pH ¼ 8: add 800 mL milliQ water to a 1 L graduated cylinder. Weigh 186.1 g of EDTA (disodium ethylenediaminetetraacetate dihydrate) and transfer to the cylinder. Adjust pH to 8 by addition of solid NaOH while stirring. EDTA will dissolve when the pH approaches 8. Make up to 1 L with milliQ water and filter through 0.22 μm filter. (i) 100 mM PMSF (phenylmethylsulfonyl fluoride): add 90 mL isopropanol to a 100 mL glass bottle with magnetic stirrer. Weigh 1.74 g PMSF and transfer to the bottle, make up to 100 mL. Dissolve by stirring and store at 20  C. (j) 1 M BZA (benzamidine): weigh 174. 63 mg of benzamidine hydrochloride monohydrate into a 15 mL falcon tube. Make up to 10 mL milliQ water and sterile filter using a syringe. Store at 20  C. (k) 500 mM glycine: add 40 mL milliQ water to a 50 mL falcon tube. Weigh 1.88 g glycine and transfer to the tube. Dissolve by vortexing and make up to 50 mL with milliQ water. (l) 100 mM iodoacetamide (IAA): weigh 0.185 g IAA and transfer to a 15 mL falcon tube. Add 10 mL milliQ water and dissolve by vortexing. (m) 1 M HEPES–NaOH: add 500 mL milliQ water to a 1 L graduated cylinder. Weigh 238.3 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

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and transfer to the cylinder. Dissolve by stirring and adjust pH to 7.5. Fill to 1 L and filter through 0.22 μm filter. (n) 1 M MgCl2: add 800 mL milliQ water to a 1 L graduated cylinder. Weigh 203.3 g of MgCl2·6H2O and transfer to the cylinder. Dissolve by stirring and fill to 1 L with milliQ water. Filter through 0.22 μm filter. 2. No attempt is made to separate the isotypes C4A and C4B in this purification protocol. 3. Avoid using glassware until the Source 15Q step. 4. The plasma is transferred to a 2 L plastic cylinder containing a magnet and submerged in ice until the filling level of the plasma. The ice bucket containing the cylinder is placed on top of a magnetic stirrer. 5. The flow through should be collected in a plastic beaker. 6. Include a C4 marker lane to monitor the presence of the C4 α chain. 7. 0.24 of the final weight needs to be added from the PEG6000 50% w/w solution in order to obtain a 12% w/w final concentration. 8. The supernatant should be separated carefully from the pellet to avoid clogging of the filter membrane in the next step. 9. BZA absorbs UV light at 280 nm, hence it may lead to baseline oscillations. 10. For C4 amounts lower than 10 mg, a 1 mL Mono Q column may be used. In this scenario elute with a 15 mL gradient and collect 0.5 mL fractions. 11. IAA should be freshly prepared before each cleavage reaction. 12. Nb5 and Nb10 are two nanobodies, the antigen binding domains of heavy chain only antibodies produced by immunization of a llama with human C4, followed by in vitro selection using phage display technology [20].

Acknowledgments We would like to thank Karen Margrethe Nielsen for invaluable technical assistance. Gregers R Andersen was supported by the BRAINSTRUC Lundbeck Foundation center, Danscatt, the Danish Research Council for independent research, and the Novo Nordisk Foundation.

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11. Escudero-Esparza A, Kalchishkova N, Kurbasic E, Jiang WG, Blom AM (2013) The novel complement inhibitor human CUB and sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly. FASEB J 27 (12):5083–5093. https://doi.org/10.1096/ fj.13-230706 12. Gaboriaud C, Frachet P, Thielens NM, Arlaud GJ (2011) The human c1q globular domain: structure and recognition of non-immune self ligands. Front Immunol 2:92. https://doi. org/10.3389/fimmu.2011.00092 13. Kjaer TR, Thiel S, Andersen GR (2013) Toward a structure-based comprehension of the lectin pathway of complement. Mol Immunol 56(4):413–422. https://doi.org/10. 1016/j.molimm.2013.05.007 14. Carroll MC, Fathallah DM, Bergamaschini L, Alicot EM, Isenman DE (1990) Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A. Proc Natl Acad Sci U S A 87(17):6868–6872 15. Sekar A, Bialas AR, de Rivera H, Davis A, Hammond TR, Kamitaki N, Tooley K, Presumey J, Baum M, Van Doren V, Genovese G, Rose SA, Handsaker RE, Daly MJ, Carroll MC, Stevens B, SA MC, Schizophrenia Working Group of the Psychiatric Genomics C (2016) Schizophrenia risk from complex variation of complement component 4. Nature 530 (7589):177–183. https://doi.org/10.1038/ nature16549 16. Xue X, Wu J, Ricklin D, Forneris F, Di Crescenzio P, Schmidt CQ, Granneman J, Sharp TH, Lambris JD, Gros P (2017) Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses. Nat Struct Mol Biol 24 (8):643–651. https://doi.org/10.1038/ nsmb.3427 17. Mortensen S, Jensen JK, Andersen GR (2016) Solution structures of complement C2 and its C4 complexes propose pathway-specific mechanisms for control and activation of the complement Proconvertases. J Biol Chem 291 (32):16494–16507. https://doi.org/10. 1074/jbc.M116.722017 18. Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, Rees I, Ludtke SJ (2007) EMAN2: an extensible image processing suite for

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electron microscopy. J Struct Biol 157 (1):38–46. https://doi.org/10.1016/j.jsb. 2006.05.009 19. Fernandez-Leiro R, Scheres SHW (2017) A pipeline approach to single-particle processing in RELION. Acta Crystallogr D Struct Biol 73

(Pt 6):496–502. https://doi.org/10.1107/ S2059798316019276 20. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797. https://doi.org/10.1146/ annurev-biochem-063011-092449

INDEX A Affinity chromatography........................22, 25, 123, 124, 131, 209, 210, 257, 258 Age-related macular degeneration (AMD)...............6, 10, 70, 163 AH50 .............................................. 12, 22, 27, 28, 30, 31 Alternative pathways ............................2, 5, 6, 22, 27, 28, 30, 70, 77, 83–94, 97, 134, 142, 147, 161, 180 Alzheimer ........................................................... 5, 34, 161 Anaphylatoxins ........................................ 2, 21, 51, 69, 84 Angioedema.................................................................7, 11 Anti-A08 of C1q .................................................. 108–112 Antibody therapies .......................................................... 21 Anti-C1-INH ....................................................... 116, 117 Anti-C3................................................................. 133, 137 Anti-C3b......................................... 4, 133–135, 137, 138 Anti-ficolins ......................................... 122–124, 126–128 Anti-ficolin-2 ........................................................ 126, 128 Anti-ficolin-3 ........................................................ 121–131 AP50 ..........................................................................30, 85 Atypical hemolytic uremic syndrome (aHUS) ...........3, 4, 7, 11, 13, 70, 84, 85, 98, 101–103, 141, 163 Autoantibodies ..........................4, 34, 71, 79, 85, 86, 93, 107, 115, 116, 121, 122, 128, 131, 133–138, 141–144, 147 Autofluorescence......................................... 184, 188, 189 Autoimmune diseases ................... 3, 10, 11, 21, 33, 107, 122, 133 Avidity.........................................207, 213, 215, 216, 222

C C1 ............................................7, 9, 10, 21, 35, 115–119, 250, 252, 257 C1 inhibitor (C1-INH) ....................7, 35, 115–117, 252 C1-inhibitor deficiency ...............................................9, 10 C1q .............................2, 4–6, 23, 26, 28, 30, 31, 33–40, 103, 107–110, 122, 180, 191, 193–200, 202, 228, 230, 238–241, 246 C1r .........................................................70, 115, 249, 250 C1s .......................... 8, 70, 115, 249, 250, 252, 254, 257 C2 ........................................................2, 4, 250, 258, 259 C3 ........................ 1–7, 9, 10, 12–14, 35, 43, 44, 51, 55, 58, 69, 70, 73, 74, 77, 78, 83–86, 97–104, 133, 134, 137, 141, 142, 147–157, 164, 180, 181, 183, 191, 196, 228, 230, 249, 250

C3a................................2, 5, 51–59, 69, 83, 84, 97, 133, 134, 137, 142 C3b ........................ 2, 6, 14, 44, 69, 70, 83–85, 97, 103, 133–135, 137, 142, 149–156, 161, 250 C3 convertase ............................2, 13, 14, 58, 70, 73, 74, 77, 78, 84, 85, 97, 134, 142, 148, 250 C3d ......................................... 14, 45, 70, 133, 134, 137, 193–197, 202 C3 deposition..................................................... 5, 97–104 C3dg ..................................................6, 14, 43, 44, 46–48 C3 glomerulopathy (C3G).......... 84, 134, 142, 147, 180 C3 nephritic factor (C3Nef)........ 4, 13, 67, 93, 147–157 C4 ...............................2, 4–6, 12, 35, 43, 103, 180, 181, 183, 191, 196, 249–262 C4d ....................... 2, 180, 191, 193–198, 200–202, 250 C5 .................2, 3, 7–9, 13, 51, 83–87, 93, 94, 141, 249 C5a..............................................2, 5, 12, 69, 83, 84, 142 C5b-9....................................................... 1, 3, 12, 84, 101 Cancers .............................. 5, 10, 33, 191, 192, 203, 235 Cell imaging .................................................................... 97 Cerebrospinal fluid (CSF) .......................... 34, 36, 38, 40 CFH ................................... 4, 70, 85, 159, 161–163, 169 CFHR1-5......................................................161–163, 169 CH50...............................12, 22, 27, 30, 31, 64, 66, 152 Classical pathway ......................... 2–5, 22, 27, 30, 34, 97, 107, 180, 249 Complements ......................1–14, 21–33, 35, 41, 43–45, 51, 53, 55, 56, 62, 69, 70, 72, 83–94, 97, 98, 100–103, 107, 115, 121, 122, 134, 141–144, 147, 149, 151, 159–162, 175, 180, 181, 187, 191, 192, 205, 228, 230, 249–262 activation ......................... 2–8, 11–14, 21, 22, 25, 28, 33, 34, 43, 44, 51–59, 70, 79, 83, 84, 87, 89, 91, 93, 101, 103, 141, 159–176, 180, 191, 206 biomarkers ................................................................... 6 cascade ................ 4, 21, 98, 134, 179, 180, 202, 249 diagnostics ...........................................................43, 88 functional activities.............................................. 61–67 hemolytic tests............................. 7, 12, 61–67, 83–95 mediated diseases .................................................... 180 proteins ............................ 1–3, 5, 12, 21, 33–40, 179, 180, 191, 192, 227–235, 237–246 therapeutics ...............................................v, 8–10, 251 Complement system (CS) ......................... 1–3, 7, 21, 28, 33, 43, 51, 61, 69, 83, 85, 97, 98, 102–104, 115, 121, 122, 147, 179, 250

Lubka T. Roumenina (ed.), The Complement System: Innovative Diagnostic and Research Protocols, Methods in Molecular Biology, vol. 2227, https://doi.org/10.1007/978-1-0716-1016-9, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Convertases ................................... 2, 4, 7, 13, 58, 83–94, 141, 142, 144, 147, 156, 250, 251 Copy number variations (CNVs) .............. 160–162, 164, 166, 169–175

D Decay of C3 convertase .................................................. 77

E Eculizumab......................7, 13, 87, 88, 90, 93, 101, 102 Endothelial cells .....................70, 97–104, 115, 192, 228 Enzyme linked immunosorbent assays (ELISA) ....22–24, 26, 30, 31, 33–40, 43–48, 51, 52, 56, 107–113, 115–138, 141–144, 148

F Factor H (FH)......................................2, 4, 6, 13, 66, 67, 69–80, 84, 85, 134, 161, 164, 239 Factor I (FI) ..........................................2, 14, 70, 84, 250 Ficolins........................................... 2, 121, 122, 124–126, 130, 131, 205–225, 250 Ficolin-2 .................................... 122–126, 129, 131, 205, 207, 209, 210, 212, 213, 215–222, 224 Ficolin-3 .................................... 122–126, 128, 129, 131, 205, 210, 212–217, 219, 221–224 Flow cytometry .......................................................97–104 Formalin-fixed paraffin-embedded (FFPE) ............................ 180–183, 187, 189, 191

AND

RESEARCH PROTOCOLS

K Kinetic measurements .......................................... 232–233 Kinetics ............................... 85, 206, 207, 213, 215–220, 224, 229, 231, 233, 237–246

L Lectin pathway (LP) ..............................2, 3, 84, 97, 104, 121, 180, 206, 250 Linear epitope ............................................................... 108 Lupus nephritis (LN)................................. 107, 128, 134, 147, 157, 180

M Membrane attack complex (MAC) .................... 1–3, 5, 6, 21, 61, 84, 87, 94 Microscopy ....................4, 101, 179, 180, 187, 206, 259 Molecular interactions .................................................. 224 Mouse kidneys...................................................... 179–189 Multiplex ................................................ 33–40, 162, 169, 198, 200, 202 Multiplex ligation-dependent probe amplification (MLPA)...............................................13, 159–176 Multivalency .................................................................. 246

N

Gel filtration .................................................................. 257 Genetic abnormalities ........................................ 13, 14, 79

Negative stain electron microscopy (NSEM) .... 251, 253, 258–260 Neoepitopes................................ 12–14, 51, 85, 141, 147 Nephelometry ...........................................................33–41 Nephritic factors..................................4, 13, 85, 141, 147 Next-generation sequencing (NGS) ........... 13, 161, 162, 164, 166, 169, 171, 172, 174, 175, 180, 187

H

P

Heme .............................................. 98, 99, 103, 227–235

Plasma ........................................7, 12, 13, 33, 34, 38, 40, 44–47, 53–56, 58, 63, 70, 74, 76–79, 98, 103, 109–112, 115, 134–136, 143, 144, 150, 151, 153, 154, 157, 161, 228, 251, 253, 256, 262 Protein–protein interactions................................ 237, 238

G

I IgG..............................21–31, 34, 45, 46, 100, 107–110, 115, 117–119, 124, 128, 134, 135, 137, 138, 142–144, 150, 151, 153–157, 181, 183, 193, 228, 230, 231, 238, 240, 241, 245, 246 IgG depletion .................................................................. 30 IgM ................................. 21–31, 34, 117, 118, 138, 144, 228, 240, 241, 245 IgM depletion ............................................. 22, 23, 25, 28 Immunoassays .................................................... 12, 33–35 Immunofluorescence (IF)...................... 14, 97, 179–189, 191–203 Immunoglobulins (Igs) .........................39, 87, 115, 116, 130, 144, 194, 227–235 Immunohistochemistry (IHC)......................14, 191–202 Ion exchange ........................................................ 209, 210

R Rabbit erythrocytes ........................ 30, 85, 86, 88–90, 94 RCA cluster .......................................................... 159–176 Recognition specificities ...................................... 205–225 Rocket immunoelectrophoresis (RIE).............. 33–40, 44

S Serum....................................... 12, 13, 21–31, 36, 38–40, 45–47, 53, 55, 56, 58, 62–64, 72–74, 77, 85–88, 90–94, 98–101, 103, 109, 110, 112, 115–117, 121, 122, 127–129, 134–137, 142, 144, 147, 150–152, 155–157, 180, 187, 192, 208

THE COMPLEMENT SYSTEM: INNOVATIVE DIAGNOSTIC Sheep erythrocytes ............................... 30, 61–64, 72–73, 75–78, 148–157 Small angle x-ray scattering .......................................... 251 Spectroscopy......................................................... 229–232 Structural variations ...................................................... 160 Surface plasmon resonance (SPR)...................... 138, 144, 205–225, 229, 238, 239, 243 Systemic lupus erythematosus (SLE).............3, 4, 11, 34, 43, 107, 108, 121, 122, 128, 133, 134, 141, 163

AND

RESEARCH PROTOCOLS Index 267

Time-resolved immunofluorometric assay (TRIFMA) .....................................................43–49 Tumors ............................................................. 5, 191–202

U UV-Vis spectroscopy............................................ 229–232

X X-ray crystallography .................................. 252, 257, 258

T Thermodynamics.................................................. 237–246